Activated Formylglycine-Generating Enzymes and Methods of Producing and Using the Same

ABSTRACT

The present disclosure provides activated formylglycine-generating enzymes (FGE), methods of producing activated FGE, and their use in methods of producing a protein comprising a formylglycine (FGly) residue. The methods of producing activated FGE, as well as methods of use of activated FGE in producing FGly-containing proteins, include both cell-based and cell-free methods. Compositions and kits that find use, e.g., in practicing the methods of the present disclosure are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/112,422, filed Feb. 5, 2015, and U.S. ProvisionalPatent Application No. 62/134,461, filed Mar. 17, 2015, whichapplications are incorporated herein by reference in their entireties.

INTRODUCTION

The properties of therapeutic proteins can be enhanced by site-specificprotein conjugation. Recombinant proteins expressed in mammalian cellscan be site-specifically modified via one or more genetically encodedaldehyde groups. For example, a peptide sequence recognized by theendoplasmic reticulum (ER)-resident formylglycine generating enzyme(FGE), which can be as short as 5 residues, may be genetically encodedinto heterologous proteins expressed in mammalian cells. FGEco-translationally converts a cysteine or serine residue of the FGErecognition site to a formylglycine residue, thereby producing proteinsbearing a unique aldehyde group. This aldehyde group may be utilized forsite-specific conjugation of an agent of interest (e.g., a therapeuticagent, an imaging agent, etc.) to the protein.

SUMMARY

The present disclosure provides activated formylglycine-generatingenzymes (FGE), methods of producing activated FGE, and their use inmethods of producing a protein comprising a formylglycine (FGly)residue. The methods of producing activated FGE, as well as methods ofuse of activated FGE in producing FGly-containing proteins, include bothcell-based and cell-free methods, Compositions and kits that find use,e.g., in practicing the methods of the present disclosure are alsoprovided.

Aspects of the present disclosure include a method of producing aprotein comprising a formylglycine residue. The method includescombining an activated formylglycine-generating enzyme (FGE) with aprotein comprising an FGE recognition site under conditions in which theactivated FGE converts a cysteine residue or a serine residue of the FGErecognition site to a formylglycine residue, to produce a proteincomprising a formylglycine residue.

In some embodiments, the combining includes culturing a cell thatincludes a formylglycine-generating enzyme (FGE), and the protein havingthe FGE recognition site, in a cell culture medium comprising Cu²⁺ undercell culture conditions in which the FGE converts the cysteine residueor the serine residue of the FGE recognition site to the formylglycineresidue.

In some embodiments, the Cu²⁺ is present in the cell culture medium at aconcentration of from 1 nM to 10 mM.

In some embodiments, the Cu²⁺ is present in the cell culture medium at aconcentration of from 1 μM to 1 mM.

In some embodiments in which the method of producing a proteincomprising a formylglycine residue involve a cell, the FGE is endogenousto the cell and/or the cell is genetically modified to express an FGE,and the protein containing an FGE recognition site is endogenous to thecell and/or the cell is genetically modified to express the proteincontaining an FGE recognition site. Either or both of the FGE and theprotein containing an FGE recognition site may be endogenous to thecell, or the cell may be genetically modified to express either or bothof the FGE and the protein containing an FGE recognition site. Where thecell is genetically modified to express an FGE, the cell may alsoexpress an FGE endogenous to the cell.

In some embodiments, the cell is a eukaryotic cell.

In some embodiments, the eukaryotic cell is a mammalian cell.

In some embodiments, the mammalian cell is selected from: a CHO cell, aHEK cell, a BHK cell, a COS cell, a Vero cell, a Hela cell, an NIH 3T3cell, a Huh-7 cell, a PC12 cell, a RAT1 cell, a mouse L cell, an HLHepG2cell, an NSO cell, a C127 cell, a hybridoma cell, a PerC6 cell, a CAPcell, and a Sp-2/0 cell.

In some embodiments, the mammalian cell is a human cell.

In some embodiments, the eukaryotic cell is a yeast cell.

In some embodiments, the eukaryotic cell is an insect cell.

In some embodiments, the combining includes expressing an FGE and theprotein comprising the FGE recognition site in a cell-free reactionmixture that includes Cu²⁺ under conditions in which the FGE convertsthe cysteine residue or the serine residue of the FGE recognition siteto the formylglycine residue.

In some embodiments, the activated FGE and the protein having the FGErecognition site are combined in a reaction mixture that includes areducing agent. In some embodiments, the reducing agent promotesconversion of the cysteine residue or the serine residue of the FGErecognition site to the formylglycine residue. In some embodiments, thereducing agent is 2-mercaptoethanol.

In some embodiments, the activated FGE and the protein having the FGErecognition site are combined in a cell-free reaction mixture.

In some embodiments, prior to combining the activated FGE with theprotein having the FGE recognition site, the method includes activatingan FGE with Cu²⁺.

In some embodiments, elemental oxygen is present as a terminal oxidant.

In some embodiments, the elemental oxygen is provided by oxygen, amixture of oxygen and hydrogen sulfide, or oxygen under basicconditions.

In some embodiments, the elemental oxygen is a terminal oxidant in areaction catalyzed by Cu²⁺.

In some embodiments, the Cu²⁺ is provided by a source of Cu²⁺ selectedfrom copper sulfate, copper citrate, copper tartrate, Fehling's reagent,and Benedict's reagent.

In some embodiments, the source of Cu²⁺ is copper sulfate.

In some embodiments, when the activated FGE and the protein having theFGE recognition site are combined in a cell-free reaction mixture, theactivated FGE is an N-terminally truncated FGE. The N-terminallytruncated FGE may be an N-terminally truncated human FGE.

In some embodiments, the protein is an antibody or antibody fragment.

In some embodiments, the antibody or antibody fragment is selected from:an IgG or fragment thereof, a Fab, a F(ab′)2, a Fab′, an Fv, an ScFv, abispecific antibody or fragment thereof, a diabody or fragment thereof,a chimeric antibody or fragment thereof, a monoclonal antibody orfragment thereof, a humanized antibody or fragment thereof, and a fullyhuman antibody or fragment thereof.

In some embodiments, the antibody specifically binds to atumor-associated antigen or a tumor-specific antigen.

In some embodiments, the tumor associated antigen or tumor-specificantigen is selected from: HER2, CD19, CD22, CD30, CD33, CD56,CD66/CEACAM5, CD70, CD74, CD79b, CD138, Nectin-4, Mesothelin,Transmembrane glycoprotein NMB (GPNMB), Prostate-Specific MembraneAntigen (PSMA), SLC44A4, CA6, and CA-IX.

In some embodiments, the protein is a ligand.

In some embodiments, the ligand is a growth factor.

In some embodiments, the ligand is a hormone.

In some embodiments, the method further includes conjugating an agent tothe protein having the formylglycine residue via an aldehyde moiety ofthe formylglycine residue.

In some embodiments, the agent is a therapeutic agent.

In some embodiments, the therapeutic agent is selected from: a cytotoxicagent, an antiproliferative agent, an antineoplastic agent, anantibiotic agent, an antifungal agent, and an antiviral agent.

In some embodiments, the agent is an imaging agent.

In some embodiments, the imaging agent is selected from: a fluorescentdye, a near-infrared (NIR) imaging agent, and a single-photon emissioncomputed tomography (SPECT)/CT imaging agent, a nuclear magneticresonance (NMR) imaging agent, a magnetic resonance imaging (MRI) agent,a positron-emission tomography (PET) agent, an x-ray imaging agent, acomputed tomography (CT) imaging agent, a K-edge imaging agent, anultrasound imaging agent, a photoacoustic imaging agent, an acousticoptical imaging agent, microwave imaging agent, a nuclear imaging agent,and combinations thereof.

Aspects of the present disclosure include a composition that includes acell culture medium that includes Cu²⁺, and a cell present in the cellculture medium, where the cell expresses formylglycine-generating enzyme(FGE).

In some embodiments, the Cu²⁺ is present in the cell culture medium at aconcentration of from 0.1 μM to 10 mM.

In some embodiments, the Cu²⁺ is present in the cell culture medium at aconcentration of from 1 μM to 1 mM.

In some embodiments in which the composition includes a cell, the FGE isendogenous to the cell and/or the cell is genetically modified toexpress an FGE, and the protein containing an FGE recognition site isendogenous to the cell and/or the cell is genetically modified toexpress the protein containing an FGE recognition site. Either of bothof the FGE and the protein containing an FGE recognition site may beendogenous to the cell, or the cell may be genetically modified toexpress either or both of the FGE and the protein containing an FGErecognition site. Where the cell is genetically modified to express anFGE, the cell may also express an FGE endogenous to the cell.

In some embodiments, the cell is a eukaryotic cell.

In some embodiments, the eukaryotic cell is a mammalian cell.

In some embodiments, the mammalian cell is selected from: a CHO cell, aHEK cell, a BHK cell, a COS cell, a Vero cell, a Hela cell, an NIH 3T3cell, a Huh-7 cell, a PC12 cell, a RAT1 cell, a mouse L cell, an HLHepG2cell, an NSO cell, a C127 cell, a hybridoma cell, a PerC6 cell, a CAPcell, and a Sp-2/0 cell.

In some embodiments, the eukaryotic cell is a yeast cell.

In some embodiments, the eukaryotic cell is an insect cell.

In some embodiments, the cell is a prokaryotic cell.

Aspects of the present disclosure include a method that includesculturing a cell that includes a nucleic acid encoding aformylglycine-generating enzyme (FGE) in a cell culture medium that hasCu²⁺, where the culturing is under conditions in which the FGE isexpressed in the cell.

In some embodiments, the Cu²⁺ is present in the cell culture medium at aconcentration of from 0.1 μM to 10 mM.

In some embodiments, the Cu²⁺ is present in the cell culture medium at aconcentration of from 1 μM to 1 mM.

In some embodiments, the cell is a eukaryotic cell.

In some embodiments, the eukaryotic cell is a mammalian cell.

In some embodiments, the mammalian cell is selected from: a CHO cell, aHEK cell, a BHK cell, a COS cell, a Vero cell, a Hela cell, an NIH 3T3cell, a Huh-7 cell, a PC12 cell, a RAT1 cell, a mouse L cell, an HLHepG2cell, an NSO cell, a C127 cell, a hybridoma cell, a PerC6 cell, a CAPcell, and a Sp-2/0 cell.

In some embodiments, the eukaryotic cell is a yeast cell.

In some embodiments, the eukaryotic cell is an insect cell.

In some embodiments, the cell is a prokaryotic cell.

Aspects of the present disclosure include a method of producing anactivated formylglycine-generating enzyme (FGE), where the methodincludes treating an FGE with Cu²⁺ to produce an activated FGE.

In some embodiments, treating the FGE with Cu²⁺ includes culturing acell that has a nucleic acid encoding the FGE in a cell culture mediumthat has Cu²⁺, where the culturing is under conditions in which the FGEis expressed in the cell.

In some embodiments, the Cu²⁺ is present in the cell culture medium at aconcentration of from 0.1 μM to 10 mM.

In some embodiments, the Cu²⁺ is present in the cell culture medium at aconcentration of from 1 μM to 1 mM.

In some embodiments, the cell is a eukaryotic cell.

In some embodiments, the eukaryotic cell is a mammalian cell.

In some embodiments, the mammalian cell is selected from: a CHO cell, aHEK cell, a BHK cell, a COS cell, a Vero cell, a Hela cell, an NIH 3T3cell, a Huh-7 cell, a PC12 cell, a RAT1 cell, a mouse L cell, an HLHepG2cell, an NSO cell, a C127 cell, a hybridoma cell, a PerC6 cell, a CAPcell, and a Sp-2/0 cell.

In some embodiments, the eukaryotic cell is a yeast cell.

In some embodiments, the eukaryotic cell is an insect cell.

In some embodiments, the cell is a prokaryotic cell.

In some embodiments, the method further includes purifying the FGE fromthe cell.

In some embodiments, treating the FGE includes expressing the FGE in acell-free reaction mixture comprising Cu²⁺.

In some embodiments, the FGE is treated with Cu²⁺ in a cell-freereaction mixture.

In some embodiments, elemental oxygen is present as a terminal oxidant.

In some embodiments, the elemental oxygen is provided by oxygen, amixture of oxygen and hydrogen sulfide, or oxygen under basicconditions.

In some embodiments, the elemental oxygen is a terminal oxidant in areaction catalyzed by Cu²⁺.

In some embodiments, the Cu²⁺ is provided by a source of Cu²⁺ selectedfrom:

copper sulfate, copper citrate, copper tartrate, Fehling's reagent, andBenedict's reagent.

In some embodiments, the method further includes purifying the FGE fromthe Cu²⁺.

In some embodiments, when the FGE is treated with Cu²⁺ in a cell-freereaction mixture, the FGE is an N-terminally truncated FGE. In someembodiments, the FGE is an N-terminally truncated human FGE.

Aspects of the present disclosure include an activatedformylglycine-generating enzyme (FGE) produced by the method disclosedherein.

Aspects of the present disclosure include a cell-free composition thatincludes an activated formylglycine-generating enzyme (FGE), and abuffer. In some embodiments, the activated FGE included in the cell-freecomposition is an N-terminally truncated FGE (e.g., an N-terminallytruncated human FGE).

In some embodiments, the composition includes a protein having an FGErecognition site.

Aspects of the present disclosure include a kit. The kit includes anactivated formylglycine-generating enzyme (FGE), and instructions forusing the activated FGE to convert a cysteine residue or a serineresidue present in an FGE recognition site of a protein to aformylglycine residue. In some embodiments, the activated FGE includedin the kit is an N-terminally truncated FGE (e.g., an N-terminallytruncated human FGE).

Aspects of the present disclosure include a kit that includes a nucleicacid that encodes a formylglycine-generating enzyme (FGE), and Cu²⁺ or asource of Cu²⁺.

In some embodiments, the kit also includes cells suitable for expressingthe FGE encoded by the nucleic acid. Such cells may express anendogenous FGE and/or express an endogenous protein containing an FGErecognition site.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1, Panels A-B provide data showing in vivo FGE activation/increasedconversion in Cu²⁺-treated cells according to certain embodiments of thepresent disclosure.

FIG. 2 provides data showing in vivo FGE activation/increased conversionin Cu²⁺-treated cells according to certain embodiments of the presentdisclosure.

FIG. 3, Panel A shows data comparing the viable density of Cu²⁺-treatedcells and untreated cells. FIG. 3, Panel B shows data comparing theprotein titer of Cu²⁺-treated cells and untreated cells. FIG. 3, Panel Cprovides data showing in vivo FGE activation/increased conversion inCu²⁺-treated cells according to an embodiment of the present disclosure.

FIG. 4 provides data showing in vivo FGE activation/increased conversionin Cu²⁺-treated cells according to an embodiment of the presentdisclosure.

FIG. 5 provides liquid chromatography-mass spectrometry data comparingFGEs isolated from Cu²⁺-treated E. coli cells and untreated E. colicells.

FIG. 6 shows data demonstrating in vitro activation of Sc FGE usingCuSO₄, according to certain embodiments of the present disclosure.

FIG. 7, Panel A shows a graph of % conversion in the presence of Cu²⁺ orabsence of Cu²⁺ according to certain embodiments of the presentdisclosure.

FIG. 7, Panel B shows a graph of lactate consumption in the presence orabsence of Cu²⁺. FIG. 7, Panel C shows a graph of glucose consumption inthe presence or absence of Cu²⁺.

FIG. 8 provides data showing a reduced variability in conversionefficiencies for in vivo FGE activation in Cu²⁺-treated cells accordingto an embodiment of the present disclosure.

FIG. 9, Panel A, and FIG. 9, Panel B, provide data showing thatproviding Cu²⁺ in the cell culture medium resulted in similar levels ofFGE but the activity of FGE from copper cultures was significantlyhigher, according to certain embodiments of the present disclosure.

FIG. 10 shows images of purified FGE characterized by SDS-PAGEelectrophoresis. Sc-FGE and Hs-cFGE preparations yielded enzyme in goodquantity and purity. FIG. 10, panel a, Lanes show intermediate stages ofpurification of Sc-FGE as follows: 1—total soluble protein after celllysis; 2—total soluble protein after DNA precipitation with 1% w/vstreptomycin sulfate; 3—soluble protein in the flow-through recoveredfrom loading the lysate on Ni-NTA resin; 4—soluble protein in the washfractions of Ni-NTA chromatography; and 5—soluble protein in the elutionfractions of Ni-NTA chromatography. FIG. 10, panel b, shows the finalpurity of 10 production batches of Hs-cFGE expressed in Hi5 cells.

FIG. 11 shows LC/MS characterization of starting material and productpeptides. Shown are spectra of the peaks with retention t=2.1 min (FIG.11, panel a), and t=3.3 min (FIG. 11, panel b) from FIG. 14, panel a.

FIG. 12 shows graphs of reaction inhibition as a function of substratedimerization. FIG. 12, panel a, shows a graph indicating that theidentity of the C_(sub)-C_(sub) dimer was confirmed by LC-MS of a sideproduct peak that formed during in vitro conversion with FGE. FIG. 12,panel b, shows a graph indicating that addition of hydrogen peroxide toFGE reaction mixtures rapidly formed substrate dimer and inhibitedproduct formation. FIG. 12, panel c, shows a graph indicating thatproduct formation was also inhibited by the presence of Cu²⁺ in reactionmixtures. This inhibition was a result of consuming DTT (FIG. 12, paneld) and trapping substrate by dimerization (FIG. 12, panel e).

FIG. 13 shows data related to identification and monitoring of substratedisulfides formed by FGE. FIG. 13, panel a, shows LC/MS identificationof Csub-BME. FIG. 13, panel b, shows an expanded HPLC trace of an FGEreaction mixture containing BME as the stoichiometric reductant. Productformation catalyzed by FGE, in the absence of reducing agent (FIG. 13,panel c), with 2.5 mol equiv of reducing agent (FIG. 13, panel d) and,with 100 mol equiv of reducing agent (FIG. 13, panel e).

FIG. 14 presents data showing the specific activity of FGEs was measuredusing a discontinuous activity assay. FIG. 14, panel a, shows data wherea 14-amino acid peptide substrate was used to measure the kinetics ofFGE catalysis. The Cys- and fGly-containing peptides were separated byRP-HPLC, and their quantities were determined by integrating the peakareas at 215 nm. The asterisk indicates an impurity formed duringpeptide synthesis. FIG. 14, panel b, shows data indicating that theinitial velocity of the reaction (v₀) was determined by extrapolating aplot of the instantaneous reaction velocity (p/t) versus time (t) to they-axis (t=0). FIG. 14, panel c, shows a schematic indicating thatwild-type Hs-FGE contains three primary domains: an ER-directing signalsequence, an N-terminal extension that interacts with ERp44 for ERretention, and a catalytic core. FIG. 14, panel d, shows a graph showingthe specific activity of eukaryotic and prokaryotic FGEs as purifiedfrom cell culture. Full length (Hs-FGE) and the core lacking the NTE(Hs-cFGE) had similar specific activities. Despite high sequence andstructural homology with Hs-FGE, the prokaryotic Sc-FGE as produced inE. coli was less active than Hs-cFGE.

FIG. 15 show data indicating that the specific activity of FGE increasedsignificantly upon treatment with stoichiometric amounts of copper(II).FIG. 15, panel a, shows a graph indicating that, as produced in E. coli,Sc-FGE had low specific activity. Treatment of Sc-FGE with two-electronoxidants or reductants (DHAA or DTT, respectively) did not change theactivity of the enzyme. In contrast, treatment with an excess of CuSO₄followed by gel filtration to remove the Cu²⁺ significantly increasedFGE activity. FIG. 15, panel b, shows a graph indicating thatsubstoichiometric amounts of Cu²⁺ did not fully activate Sc-FGE.Addition of 1 or more molar equiv of Cu²⁺ fully activated the enzyme.FIG. 15, panel c, shows a graph indicating that, as purified, Sc-FGEcontained ˜0.01 Cu/FGE, and Hs-cFGE contained 0.34 Cu/FGE. Afteractivation in vitro, Sc-FGE and Hs-FGE contained 1.1 and 1.4 Cu/FGE,respectively. In all cases, the amount of copper contained inpreparations of purified enzyme correlated with the specific activity ofthe enzyme. FIG. 15, panel d, shows formation of the oxidized form ofDTT as well as C_(sub)-C_(sub) as a result of copper-catalyzed disulfideformation.

FIG. 16 shows data indicating that specific activity of Hs-cFGE did notcorrelate with the redox state of active site residue C₃₄₁. FIG. 16,panel a, shows a schematic of Hs-cFGE, which contains two structuraldisulfides and two active site cysteines, which can form a disulfide inthe apoenzyme. FIG. 16, panel b, shows a graph indicating that the redoxstate of C₃₄₁ can be measured by LC-MS/MS. After activation with Cu²⁺,the amount of C₃₄₁ accessible to solution was ˜28%. Upon treatment withreducing agent (DTT) the amount of accessible C₃₄₁ increased to 93%.However, the specific activity of the enzyme did not decrease. Treatmentwith DTT did not affect C₂₃₅-containing structural disulfide.

FIG. 17 shows data indicating that FGE catalysis was inhibited bycyanide. FIG. 17, panel a, shows a graph indicating that pretreatment ofactivated FGEs with EDTA or KCN resulted in little to no change inspecific activity. Only Hs-cFGE activity decreased modestly after KCNtreatment. FIG. 17, panel b, shows a graph indicating that both Sc-FGEand c, Hs-cFGE were inhibited during turnover in aconcentration-dependent manner by CN⁻ which was a strong ligand forcopper. FIG. 17, panel d, shows pretreatment of activated Sc-FGE (n=6)with reductants (DTT, TCEP) or a metal chelator (EDTA) do not change thespecific activity of the enzyme. When the reductant and chelator arecombined (15 mM each, 1 h, 37° C.), the FGE activity is nearlyeliminated.

FIG. 18 shows data of kinetic parameters of Sc-FGE and Hs-cFGE asdetermined by nonlinear regression. FIG. 18, panel a, shows aMichaelis-Menten plot correlating substrate concentration with v₀ forSc-FGE and for Hs-cFGE (FIG. 18, panel b). The data were fit to theMichaelis-Menten equation by nonlinear regression to determine thekinetic parameters (FIG. 18, panel c).

FIG. 19 shows data indicating that FGE can catalyze substrate/reductantdisulfide formation, which can be inhibitory to product yield, andconfirms that substrate was bound in the active site as a disulfide.FIG. 19, panel a, shows a graph of product formation in FGE reactionmixtures across a range of 0-10 mM βME. Without βME, fGly andC_(sub)-C_(sub) formed at comparable rates. As [βME] was increased,C_(sub)-C_(sub) was replaced by C_(sub)-βME, and product formed at ahigher rate. FIG. 19, panel b, shows a graph indicating that the initialvelocity of FGE catalysis with βME was higher than with DTT. FIG. 19,panel c, shows a schematic indicating that these data confirm thatformation of [E⋅S] resulted, i, in a disulfide between E and S.Catalysis ii, converted substrate to product, which was then releasediii. In the presence of thiol reducing agents, the rate of catalysisdefined by k_(cat) was in competition with thiol-disulfide exchange iv,with rate constant k_(DS), which dissociated the E⋅S complex andreleased C_(sub)-R. This species can then react with another equivalentof reducing agent v, to regenerate free substrate. When the reagentadded was a strong non-thiol reductant (e.g. TCEP) or a thiol that cancyclize (DTT), iv and V collapsed to a single step. When the reducingagent was a monothiol, the C_(sub)-R can persist long enough to reform[E⋅S] with rate constant k_(−DS).

FIG. 20 shows data indicating that FGE can be used to produce aldehydetagged mAbs in high yield. FIG. 20, panel a, shows a schematicindicating that the aldehyde tag was installed in three regions (hinge,CH₂, and CH₃) of the heavy chain of an IgG1 heavy chain. After reactionwith FGE in vitro, the quantities of Cys and fGly were determined. Underoptimized conditions, the yield of conversion from Cys to fGly wasconsistently 85-95%. FIG. 20, panel b, shows a graph indicating that theyield of biocatalytic fGly production was independent of reaction scale,as measured in 0.8, 8.0 and 80.0 mg test cases on two individual mAbs.FIG. 20, panel c, shows a graph of a representative example from alarger scale reaction. After conversion with FGE, the fGly content was98.1%, corresponding to a conversion yield of 97.8%.

FIG. 21 shows data related to LC-MRM/MS characterization of Cys- andfGly-containing proteins. FIG. 21, panel a, shows a schematic indicatingthat a monoclonal antibody was disassembled and digested, and the Cysand fGly functional groups were trapped as the carboxyamidomethyl (CAM)and methyl oxime (MeOx) species, respectively, for characterization,through the following steps: i) DTT, 37° C., 15 min; ii) HCl, thenNH₄HCO₃; iii) trypsin, iodoacetamide, pH 8, 37° C., 1 h; iv)methoxylamine, pH 5.5, 16 h FIG. 21, panel b, shows a graph indicatingthat each aldehyde tag location was characterized by a unique trypticpeptide containing the installed tag sequence. Here, a 9-mer peptide wasgenerated by trypsin. Targeted MRM-MS was used to quantify the peptidesmodified as CAM or MeOx. The five most intense precursor/product iontransitions were chosen for peak area integration. FIG. 21, panel cshows an ion chromatogram of the CAM- and MeOx-containing peptides. TheMeOx-containing peptide separated into the diastereomers present as aresult of oxime formation.

FIG. 22 shows data related to the biophysical properties of mAbssubjected to in vitro conversion reaction conditions. FIG. 22, panel a,and FIG. 22, panel b, shows graphs of ELISA measurements ofantibody/antigen affinity for two IgG1 mAbs. FIG. 22, panel c, shows agraph indicating that measurement of the proportion of oxidizedmethionine at position 252 in the CH₃ domain of an IgG1 scaffold wasunchanged over the course of the reaction.

FIG. 23 shows a schematic of the proposed catalytic mechanism for FGEthat accounts for the copper cofactor. As shown in step a, substrate Sbinds enzyme E and is covalently attached through C₃₄₁. This covalentbond formation may be catalyzed by Cu²⁺, or it could result fromthiol-disulfide exchange between an existing C₃₄₁ disulfide andsubstrate. As shown in step b, reduction of Cu²⁺ to Cu¹⁺ would enablebinding of molecular oxygen (step c), as the Cu²⁺ superoxo intermediate.As shown in step d, oxidation of substrate by the Cu(II)-O₂ ⁻ throughproton-coupled electron transfer (likely hydrogen atom transfer) wouldgenerate a disulfide radical, which would rapidly collapse, (step e), tothioaldehyde and thiyl radical. A second 1H⁺/1 e⁻ reduction andhydrolysis would regenerate C₃₄₁. The way in which this reduction occurscould proceed through a number of pathways, including, but not limitedto: direct thiyl radical reduction and hydrolysis of copper-peroxide torelease H₂O₂; or oxo transfer from Cu(II)-OOH to the radical to generatea sulfenic acid, followed by copper-oxyl reduction and sulfenic acidhydrolysis.

DETAILED DESCRIPTION

The present disclosure provides activated formylglycine-generatingenzymes (FGE), methods of producing activated FGE, and their use inmethods of producing a protein comprising a formylglycine (FGly)residue. The methods of producing activated FGE, as well as methods ofuse of activated FGE in producing FGly-containing proteins, include bothcell-based and cell-free methods, Compositions and kits that find use,e.g., in practicing the methods of the present disclosure are alsoprovided.

Before the methods and compositions of the present disclosure aredescribed in greater detail, it is to be understood that the methods andcompositions are not limited to particular embodiments described, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe methods and compositions will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within by the methods and compositions. Theupper and lower limits of these smaller ranges may independently beincluded in the smaller ranges and are also encompassed within by themethods and compositions, subject to any specifically excluded limit inthe stated range. Where the stated range includes one or both of thelimits, ranges excluding either or both of those included limits arealso included in the methods and compositions.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the methods belong. Although any methods andcompositions similar or equivalent to those described herein can also beused in the practice or testing of the methods and compositions,representative illustrative methods and compositions are now described.

Any publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the materials and/or methods and compositions in connectionwith which the publications are cited. The citation of any publicationis for its disclosure prior to the filing date and should not beconstrued as an admission that the present methods and compositions arenot entitled to antedate such publication, as the date of publicationprovided may be different from the actual publication date which mayneed to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the methods and compositions,which are, for clarity, described in the context of separateembodiments, may also be provided in combination in a single embodiment.Conversely, various features of the methods and compositions, which are,for brevity, described in the context of a single embodiment, may alsobe provided separately or in any suitable sub-combination. Allcombinations of the embodiments are specifically embraced by the presentdisclosure and are disclosed herein just as if each and everycombination was individually and explicitly disclosed, to the extentthat such combinations embrace operable processes and/or devices. Inaddition, all sub-combinations listed in the embodiments describing suchvariables are also specifically embraced by the present methods andcompositions and are disclosed herein just as if each and every suchsub-combination was individually and explicitly disclosed herein.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the otherembodiments without departing from the scope or spirit of the presentmethods. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Formylglycine-Generating Enzymes, Fge Recognition Sequences, and TargetProteins

Aspects of the present disclosure include methods of making activatedformylglycine-generating enzymes (FGEs), and methods of use of activatedFGEs to produce formylglycine-containing proteins by conversion of anamino acid in an FGE recognition sequence in a protein of interest.Compositions that include activated FGEs and proteins of interest, whichproteins include one or more FGE recognition sequences and/or one ormore formylglycine residues, are also provided.

Described below are examples of FGEs that may be activated for use inthe methods and compositions of the present disclosure, examples of FGErecognition sequences that may be provided in a protein of interest, andexamples of proteins of interest which may be converted by FGE toinclude a formylglycine residue useful, e.g., for conjugating an agent(e.g., a therapeutic agent, an imaging agent, etc.) to the protein ofinterest.

Formylglycine-Generating Enzymes

As used herein, a “formylglycine-generating enzyme” (or “FGE”) is anenzyme that oxidizes cysteine or serine in a sulfatase motif (or “FGErecognition site”) to a 2-formylglycine (FGly) residue (also referred toherein as a formylglycine residue). Thus, an “FGE” is used herein torefer to any enzyme that can act as an FGly-generating enzyme to mediateconversion of a cysteine (“Cys” or “C”) of an FGE recognition site toFGly or that can mediate conversion of serine (“Ser” or “S”) of an FGErecognition site to FGly. By “conversion” as used in the context ofaction of an FGE on an FGE recognition site refers to biochemicalmodification of a cysteine or serine residue in an FGE recognition siteto a formylglycine (FGly) residue (e.g., Cys to FGly, or Ser to FGly).FGE recognition sites modified by an FGE to contain an FGly may bereferred to herein as a “converted FGE recognition site”.

It should be noted that in general, the literature refers toFGly-generating enzymes that convert a Cys to FGly in an FGE recognitionsite as FGEs, and refers to enzymes that convert Ser to FGly in an FGErecognition site as Ats-B-like. However, for purposes of the presentdisclosure “FGE” is used generically to refer to any enzyme thatexhibits an FGly-generating enzyme activity at an FGE recognition site,with the understanding that an appropriate FGE may be selected accordingto the FGE recognition site (i.e., Cys-containing or Ser-containing)and/or the target protein containing the FGE recognition site.

As evidenced by the ubiquitous presence of sulfatases having an FGly atthe active site, FGEs are found in a wide variety of cell types,including both eukaryotes and prokaryotes. There are at least two formsof FGEs. Eukaryotic sulfatases generally contain a cysteine in theirsulfatase motif and are modified by the “SUMF1-type” FGE (see, e.g.,Cosma et al. Cell 2003, 113, (4), 445-56; Dierks et al. Cell 2003, 113,(4), 435-44), which may be encoded by a SUMF1 gene. Prokaryoticsulfatases can contain either a cysteine or a serine in their sulfatasemotif and are modified either by the “SUMF1-type” FGE or the “AtsB-type”FGE, respectively (see, e.g., Szameit et al. J Biol Chem 1999, 274,(22), 15375-81). Examples of prokaryotic FGEs include a Mycobacteriumtuberculosis (Mtb) FGE and a Streptomyces coelicolor FGE. FGEs are alsofound in deuterostomia, including vertebrates and echinodermata (see,e.g., Pepe et al. (2003) Cell 113, 445-456, Dierks et al. (2003) Cell113, 435-444; Cosma et al. (2004) Hum. Mutat. 23, 576-581).

In eukaryotes, FGE activity on a protein containing an FGE recognitionsequence may occur during or shortly after translation of the protein inthe endoplasmic reticulum (ER) (Dierks et al. Proc Natl Acad Sci USA1997, 94(22):11963-8). Without being bound by theory, in prokaryotes itis thought that SUMF1-type FGE functions in the cytosol and AtsB-typeFGE functions near or at the cell membrane. A SUMF2 FGE has also beendescribed in deuterostomia, including vertebrates and echinodermata(see, e.g., Pepe et al. (2003) Cell 113, 445-456, Dierks et al. (2003)Cell 113, 435-444; Cosma et al. (2004) Hum. Mutat. 23, 576-581).

An FGE for use in the methods and compositions disclosed herein can beobtained from naturally occurring sources or synthetically produced. Forexample, an appropriate FGE can be derived from biological sources whichnaturally produce an FGE or which are genetically modified to express arecombinant gene encoding an FGE. When the methods described hereininvolve use of a host cell, the FGE may be native to the host cell, orthe host cell can be genetically modified to express an FGE.Accordingly, the present disclosure also provides recombinant host cellsgenetically modified to express an FGE, which FGE may be selected so asto be compatible for use in conversion of a target protein having aselected FGE recognition site. In some embodiments, it may be desired touse a sulfatase motif compatible with a human FGE (see, e.g., theSUMF1-type FGE, see, e.g., Cosma et al. Cell 113, 445-56 (2003); Dierkset al. Cell 113, 435-44 (2003)), and express the protein having an FGErecognition site in a human cell that expresses a human FGE, or in ahost cell, usually a mammalian cell, genetically modified to express ahuman FGE.

In certain embodiments, the FGE is a eukaryotic FGE, such as, but notlimited to, a mammalian FGE. In some instances, the mammalian FGE is ahuman FGE. In certain embodiments, the FGE is a prokaryotic FGE, suchas, but not limited to, a bacterial FGE. In some instances, thebacterial FGE is a Mycobacterium tuberculosis (Mtb) FGE or aStreptomyces coelicolor (S. coelicolor) FGE.

FGEs, as well as nucleic acids encoding a number of FGEs, are known inthe art. See, for example in: Preusser et al. 2005 J. Biol. Chem.280(15):14900-10 (Epub 2005 Jan. 18) (describing human FGEs and nucleicacids encoding the same); Fang et al. 2004 J Biol Chem. 279(15):14570-8(Epub 2004 Jan. 28) (describing the bacterial formylglycine-generatingsulfatase-modifying enzyme AtsB of Klebsiella pneumonia and nucleicacids encoding the same); Landgrebe et al. Gene 2003 Oct. 16; 316:47-56(describing the identification of the gene (SUMF1) encoding human FGEand its conservation with prokaryotic genes encoding FGEs); Dierks etal. 1998 FEBS Lett. 423(1):61-5; Dierks et al. Cell. 2003 May 16;113(4):435-44 (describing the gene encoding human FGE and mutationstherein that cause Multiple Sulfatase Deficiency (MSD)); Cosma et al.(2003 May 16) Cell 113(4):445-56 (describing the gene encoding human FGEand mutations therein that cause Multiple Sulfatase Deficiency (MSD));Baenziger (2003 May 16) Cell 113(4):421-2 (review); Dierks et al. Cell.2005 May 20; 121(4):541-52; Roeser et al. (2006 Jan. 3) Proc Natl AcadSci USA 103(1):81-6; WO 2004/072275 (describing nucleic acids encodinghuman FGE and variants thereof); GenBank Accession No. NM_182760 (asingle nucleotide variant of human FGE); and Carlson et al. J Biol Chem.2008 283:20117-20125 (describing nucleic acids that encode Mycobacteriumtuberculosis (Mtb) FGE and Streptomyces coelicolor (S. coelicolor) FGE).

According to certain embodiments, the FGE is a full-length FGE. By“full-length” is meant the FGE has a complete, mature amino acidsequence of a wild-type FGE, including any wild-type isoforms (e.g., asa result of alternative splicing) of the corresponding wild-type FGE. Asone example of a full-length FGE, the FGE may be a full-length humanFGE, such as the full-length human FGE that includes an amino acidsequence having at least 80%, 85%, 90%, 95%, or more (e.g., 100%)sequence identity to the amino acid sequenceMAAPALGLVCGRCPELGLVLLLLLLSLLCGAAGSQEAGTGAGAGSLAGSCGCGTPQRPGAHGSSAAAHRYSREANAPGPVPGERQLAHSKMVPIPAGVFTMGTDDPQIKQDGEAPARRVTIDAFYMDAYEVSNTEFEKFVNSTGYLTEAEKFGDSFVFEGMLSEQVKTNIQQAVAAAPWWLPVKGANWRHPEGPDSTILHRPDHPVLHVSWNDAVAYCTWAGKRLPTEAEWEYSCRGGLHNRLFPWGNKLQPKGQHYANIWQGEFPVTNTGEDGFQGTAPVDAFPPNGYGLYNIVGNAWEWTSDWWTVHHSVEETLNPKGPPSGKDRVKKGGSYMCHRSYCYRYRCAARSQNTPDSSASNLGFR CAADRLPTMD (SEQ IDNO:1; provided in Table 4 below).

In other embodiments, the FGE is an N-terminally truncated FGE thatretains formylglycine-generating activity. By “N-terminally truncated”is meant the FGE includes fewer than the number of amino acids presentat the N-terminus of the corresponding wild-type FGE, including anywild-type isoforms (e.g., as a result of alternative splicing) of thecorresponding wild-type FGE. Whether an N-terminally truncated FGEretains formylglycine-generating activity may be determined using anyconvenient approach, including combining the truncated FGE in vitro witha protein that includes an FGE recognition site under conditions inwhich an FGE having formylglycine-generating activity would convert acysteine (or serine) residue of the FGE recognition site to aformylglycine residue, and determining whether the cysteine (or serine)residue of the FGE recognition site was converted to a formylglycineresidue. Example in vitro reaction conditions and methods for detectingconversion to a formylglycine residue are described elsewhere herein.

In certain aspects, the N-terminally-truncated FGE (e.g., a human FGEtruncated at the N-terminus) has a 1-72 amino acid N-terminaltruncation, and maybe lack 1-5 amino acids, 1-10 amino acids, 1-15 aminoacids, 1-20 amino acids, 1-25 amino acids, 1-30 amino acids, 1-35 aminoacids, 1-40 amino acids, 1-45 amino acids, 1-50 amino acids, 1-55 aminoacids, 1-60 amino acids, or 1-70 amino acids at the N-terminus relativeto the corresponding full-length FGE.

In certain aspects, the truncated FGE is a human FGE truncated at theN-terminus, where the N-terminally-truncated human FGE is from 300 to373 amino acids in length (e.g., 302 to 373 amino acids in length), suchas from 302 to 370, from 302 to 360, from 302 to 350, from 302 to 340,from 302 to 330, from 302 to 320, or from 302 to 310 amino acids inlength.

According to certain embodiments, the N-terminally truncated FGEcorresponds in length to a naturally-occurring FGE protease cleavageproduct. For example, the N-terminally truncated FGE may correspond inlength to a furin cleavage product of a human FGE. The furin enzymecleaves between the arginine at position 72 and the glutamic acid atposition 73 of human FGE, resulting in a cleavage product having a72-amino acid N-terminal truncation relative to a human FGE that is notcleaved by the furin enzyme. According to one embodiment, theN-terminally truncated human FGE corresponds to the furin cleavageproduct of a human FGE that results in a 72 amino acid N-terminaltruncation relative to a full-length human FGE, as shown in Table 4 (SEQID NO: 1).

TABLE 4Example full-length human FGE (non-underlined and underlined amino acids)and example truncated human FGE (underlined only) Full-length humanMAAPALGLVCGRCPELGLVLLLLLLSLLCGAAGSQEAGTGAG FGE (non-AGSLAGSCGCGTPQRPGAHGSSAAAHRYSREANAPGPVPG underlined andERQLAHSKMVPIPAGVFTMGTDDPQIKQDGEAPARRVTIDAF underlined aminoYMDAYEVSNTEFEKFVNSTGYLTEAEKFGDSFVFEGMLSEQV acids) (SEQ ID NO:KTNIQQAVAAAPWWLPVKGANWRHPEGPDSTILHRPDHPVL 1)HVSWNDAVAYCTWAGKRLPTEAEWEYSCRGGLHNRLFPWG N-terminallyNKLQPKGQHYANIWQGEFPVTNTGEDGFQGTAPVDAFPPNG truncated humanYGLYNIVGNAWEWTSDWWTVHHSVEETLNPKGPPSGKDRV FGE (underlinedKKGGSYMCHRSYCYRYRCAARSQNTPDSSASNLGFRCAAD amino acids only) RLPTMD(SEQ ID NO: 2)

Nucleic acids encoding a full-length FGE or a truncated FGE, as well asexpression vectors including the same, may be prepared using anysuitable approach, including a recombinant DNA-based approach. As anexample, a nucleic acid encoding a truncated FGE may be prepared byrestriction digestion of a nucleic acid encoding a full-length FGE, orPCR amplification of the region encoding the truncated FGE using anucleic acid encoding the full-length FGE as template. Such restrictiondigestion or amplification products may be cloned into an expressionvector suitable for expression of the full-length or truncated FGE in ahost cell of interest. The host cell of interest may then betransformed/transfected with the expression vector for subsequentproduction of the FGE in the host cell. Expression vectors and hostcells that find use in producing full-length and truncated FGEs aredescribed hereinbelow.

FGEs (e.g., full-length FGEs or N-terminally truncated FGEs) can beprovided as a fusion protein in which the FGE is fused to an amino acidsequence heterologous to the FGE (e.g., a purification tag, a proteaserecognition sequence, secretion signal sequence, an endoplasmicreticulum (ER)-directing signal sequence, an ER retention sequence(e.g., KDEL (Lys-Asp-Glu-Leu)), and/or the like). FGEs disclosed herein(e.g., full-length FGEs, N-terminally truncated FGEs, and/or fusionproteins thereof) may be used in the methods as disclosed herein toprovide activated FGEs.

FGE Recognition Sites

The FGE recognition site (also referred to herein as a “sulfatasemotif”) of the protein may vary. A minimal recognition site is usually 5or 6 amino acid residues in length, usually no more than 6 amino acidresidues in length. The entire recognition site provided in the proteinis at least 5 or 6 amino acid residues, and can be, for example, from 5to 16, 6-16, 5-15, 6-15, 5-14, 6-14, 5-13, 6-13, 5-12, 6-12, 5-11, 6-11,5-10, 6-10, 5-9, 6-9, 5-8, or 6-8 amino acid residues in length, so asto define an FGE recognition site of less than 16, 15, 14, 13, 12, 11,10, 9, 8, 7 or 6 amino acid residues in length. In certain embodiments,the FGE recognition site of the protein is described by the formula:

X¹Z¹X²Z²X³Z³  (I)

where

Z¹ is cysteine or serine (which can also be represented by (C/S));

Z² is either a proline or alanine residue (which can also be representedby (P/A));

Z³ is a basic amino acid (e.g., arginine (R), and may be lysine (K) orhistidine (H), usually lysine), or an aliphatic amino acid (alanine (A),glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P),usually A, G, L, V, or I;

X¹ is present or absent and, when present, can be any amino acid, thoughusually an aliphatic amino acid, a sulfur-containing amino acid, or apolar, uncharged amino acid, (i.e., other than an aromatic amino acid ora charged amino acid), usually L, M, V, S or T, more usually L, M, S orV, with the proviso that when the FGE recognition site is at theN-terminus of the target polypeptide, X¹ is present; and

X² and X³ independently can be any amino acid, though usually analiphatic amino acid, a polar, uncharged amino acid, or a sulfurcontaining amino acid (i.e., other than an aromatic amino acid or acharged amino acid), e.g., S, T, A, V, G or C; e.g., S, T, A, V or G. Inone example, the FGE recognition site of the protein is of the formulaL(C/S)TPSR (SEQ ID NO: 3), e.g., LCTPSR (SEQ ID NO: 4) or LSTPSR (SEQ IDNO: 5).

Examples of FGE recognition sites are described in, e.g., U.S. Pat. No.7,985,783 and U.S. Patent Application Publication No. US2011/0117621,the disclosures of which are incorporated herein by reference in theirentireties for all purposes.

Proteins that Include FGE Recognition Sites

The protein containing an FGE recognition site may be any protein ofinterest and includes proteins to which it is desirable to conjugate anagent of interest, e.g., a therapeutic agent, an imaging agent, etc.Proteins of interest include those having a naturally-occurring aminoacid sequence, a native amino acid sequence having an N-terminalmethionine, fragments of naturally-occurring proteins, and non-naturallyoccurring proteins and fragments thereof. In some embodiments, theprotein is a protein other than a sulfatase or fragment thereof, otherthan a reporter protein, or other than preprolactin or prolactin.

The protein containing the FGE recognition sequence can be of the sameor different origin as the FGE. Where the methods of the presentdisclosure involve a cell-based method, the protein containing the FGErecognition site may be endogenous to the host cell (e.g., a sulfatase)and/or the host cell may be genetically modified to express the proteincontaining an FGE recognition sequence. In this embodiment, the FGE maybe endogenous to the host cell and/or the host cell may be geneticallymodified to express an FGE.

In certain aspects, the protein is a protein that may provide for atherapeutic or other clinical benefit, including proteins for whichattachment to a moiety can provide for one or more of, for example,increased cytotoxicity upon binding of the protein to a target cell(e.g., a cancer cell), imaging (e.g., in vivo imaging) of a cell towhich the protein binds, an increase in serum half-life, a decrease inan adverse immune response, additional or alternate biological activityor functionality, or other benefit or reduction of an adverse sideeffect. Where the therapeutic protein is an antigen for a vaccine,modification can provide for an enhanced immunogenicity of the protein.

The protein may be a member of a class of proteins, such as therapeuticproteins, including, but not limited to, cytokines, chemokines, ligands,growth factors, hormones, growth hormones, enzymes (e.g., sulfatases,e.g., a human sulfatase or functional fragment thereof), antibodies andantibody fragments (including antigen-binding antibody fragments), andantigens. Further examples include erythropoietin (EPO, e.g., nativeEPO, synthetic EPO (see, e.g., US 2003/0191291), human growth hormone(hGH), bovine growth hormone (bGH), follicle stimulating hormone (FSH),interferon (e.g., IFN-gamma, IFN-beta, IFN-alpha, IFN-omega, consensusinterferon, and the like), insulin, insulin-like growth factor (e.g.,IGF-I, IGF-II), blood factors (e.g., Factor VIII, Factor IX, Factor X,tissue plasminogen activator (TPA), and the like), colony stimulatingfactors (e.g., granulocyte-CSF (G-CSF), macrophage-CSF (M-CSF),granulocyte-macrophage-CSF (GM-CSF), and the like), transforming growthfactors (e.g., TGF-beta, TGF-alpha), interleukins (e.g., IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-12, and the like), epidermalgrowth factor (EGF), platelet-derived growth factor (PDGF), fibroblastgrowth factors (FGFs, e.g., aFGF, bFGF), glial cell line-derived growthfactor (GDNF), nerve growth factor (NGF), RANTES, and the like.

In some embodiments, the protein may provide a scaffold for attachmentof a moiety of interest, such as a drug and/or imaging agent. Examplesof such proteins include, but are not limited to, serum albumin (e.g.,human serum albumin, bovine serum albumin), an Fc polypeptide (e.g., IgGFc fragment (e.g., IgG1 Fc fragment, IgG2 Fc fragment, IgG3 Fc fragment,or IgG4 Fc fragment)), and the like. Here the protein is an Fcpolypeptide, the Fc polypeptide may be a mammalian Fc polypeptide (e.g.,human Fc polypeptide).

According to certain embodiments, the protein is an antibody or anantibody fragment. The terms “antibody” and “immunoglobulin” includeantibodies or immunoglobulins of any isotype, whole antibodies (e.g.,antibodies composed of a tetramer which in turn is composed of twoheterodimers of a heavy and light chain polypeptide, including wholeIgG, IgA, IgD, IgE, or IgM antibodies); half antibodies (e.g.,antibodies that include a single dimer of a heavy and light chainpolypeptide); antibody fragments (e.g., fragments of whole antibodies,such as fragments of IgG, IgA, IgD, IgE, or IgM antibodies) which retainspecific binding to an antigen of interest, including, but not limitedto Fab, F(ab′)2, Fab′, Fv, scFv, bispecific antibodies and diabodies;chimeric antibodies; monoclonal antibodies; humanized antibodies (e.g.,humanized monoclonal antibodies, or humanized antibody fragments); orfully human antibodies (an antibody that comprises human immunoglobulinprotein sequences only). Also included are human monoclonal antibodiesthat possess somatic mutations and/or N- or P-nucleotide additions anddeletions as a result of V-D-J rearrangement. Also included are humanantibodies to which synthetic sequences have been inserted into thecomplementarity determining regions (CDRs) (see, e.g., Miersch S & SidhuSS (2012) Synthetic antibodies: concepts, potential and practicalconsiderations. Methods 57(4):486-98; and Knappik et al. (2000) Fullysynthetic human combinatorial antibody libraries (HuCAL) based onmodular consensus frameworks and CDRs randomized with trinucleotides. J.Mol. Biol. 296(1):57-86). In certain aspects, an antibody of the presentdisclosure is selected from an IgG (e.g., an IgG1, IgG2, IgG3 or IgG4antibody), Fab, F(ab′)2, and Fab′.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an F(ab′)2 fragment thathas two antigen combining sites and is still capable of cross-linkingantigen.

“Fv” comprises the minimum antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy- and one light-chain variable domain in tight,non-covalent association. It is in this configuration that the threeCDRs of each variable domain interact to define an antigen-binding siteon the surface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The “Fab” fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxyl terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)2 antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of an antibody, where these domains are present in asingle polypeptide chain. In some embodiments, the Fv polypeptidefurther comprises a polypeptide linker between the V_(H) and V_(L)domains, which enables the sFv to form the desired structure for antigenbinding.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites.

The term “recombinant” antibody as used herein is intended to includeall antibodies that are prepared, expressed, created, or isolated byrecombinant means, such as (i) antibodies expressed using a recombinantexpression vector transfected into a host cell; (ii) antibodies isolatedfrom a recombinant, combinatorial antibody library; (iii) antibodiesisolated from an animal (e.g. a mouse) that is transgenic for humanimmunoglobulin genes; or (iv) antibodies prepared, expressed, created,or isolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences, including, forexample, in-vitro translation technology (see, e.g., Yin et al. (2012) Aglycosylated antibodies and antibody fragments produced in a scalable invitro transcription-translation system, Landes Bioscience, Volume 4,Issue 2). Such recombinant antibodies include humanized, CDR grafted,chimeric, deimmunized, and in vitro generated antibodies; and canoptionally include constant regions derived from human germlineimmunoglobulin sequences.

By “humanized antibody” is meant immunoglobulins, half antibodies,immunoglobulin chains (e.g., a light chain polypeptide) or fragmentsthereof (such as Fv, scFv, Fab, Fab′, F(ab′)2 or other antigen-bindingsubsequences of antibodies) which contain sequence derived from bothhuman and non-human immunoglobulin. The humanized antibodies may behuman immunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat, lama, camel or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, a humanized antibody may comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences.

Human light chain polypeptides are typically classified as kappa andlambda light chains. Furthermore, human heavy chain polypeptides aretypically classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.Within light and heavy chains, the variable and constant regions arejoined by a “J” region of about 12 or more amino acids, with the heavychain also including a “D” region of about 10 more amino acids.

In certain aspects, the protein comprising an FGE recognition site is anIgG or fragment thereof, a Fab, a F(ab′)2, a Fab′, an Fv, an ScFv, abispecific antibody or fragment thereof, a diabody or fragment thereof,a chimeric antibody or fragment thereof, a monoclonal antibody orfragment thereof, a humanized antibody or fragment thereof, and a fullyhuman antibody or fragment thereof.

Antigens of interest to which the antibody specifically binds includetumor-specific antigens, e.g., antigens present on the surface ofmalignant cells and not present on non-malignant cells. In otheraspects, the antigen bound by the antibody is a tumor-associatedantigen. By “tumor-associated antigen” is meant an antigen expressed onmalignant cells with limited expression on cells of normal tissues,antigens that are expressed at much higher density on malignant versusnormal cells, or antigens that are developmentally expressed.

Any tumor-associated antigen or tumor-specific antigen may be targetedby an antibody of the present disclosure. In certain aspects, when themethods of the present disclosure are for treatment of cancer, theantigen specifically bound by the antibody or antibody component of aconjugate of the present disclosure may include, but is not limited to,HER2, CD19, CD22, CD30, CD33, CD56, CD66/CEACAM5, CD70, CD74, CD79b,CD138, Nectin-4, Mesothelin, Transmembrane glycoprotein NMB (GPNMB),Prostate-Specific Membrane Antigen (PSMA), SLC44A4, CA6, CA-IX, or anyother tumor-associated or tumor-specific antigens of interest.

By “specific binding” or “specifically binds” in the context of acharacteristic of an antibody refers to the ability of an antibody topreferentially bind to a particular antigen that is present in ahomogeneous mixture of different antigens.

In certain embodiments, a specific binding interaction will discriminatebetween desirable and undesirable antigens (or “target” and “non-target”antigens) in a sample or organism (e.g., a human), in some embodimentsmore than about 10 to 100-fold or more (e.g., more than about 1000- or10,000-fold). In certain embodiments, the affinity between an antibodyand antigen when they are specifically bound in an antibody-antigencomplex is characterized by a KD (dissociation constant) of less than10⁻⁶M, less than 10⁻⁷M, less than 10⁻⁸M, less than 10⁻⁹M, less than10⁻¹⁰ M, less than 10⁻¹¹M, or less than about 10⁻¹² M or less.

Methods of Producing Activated FGEs

The present disclosure provides methods of producing activated FGEs. Asused herein, an “activated formylglycine-generating enzyme” or“activated FGE” refers to an FGE that has been treated with an oxidationreagent by addition of oxidation reagent to a cell expressing an FGEand/or by addition of oxidation reagent to isolated FGE. Accordingly, incertain aspects, the present disclosure provides methods of producing anactivated FGE, which methods include treating an FGE with an oxidationreagent to produce an activated FGE. By “oxidation reagent” is meant areagent capable of oxidizing FGE (which may be referred to as an“oxidizing agent”), a reagent capable of catalyzing the oxidation ofFGE, or a combination thereof. One example of a reagent capable ofcatalyzing the oxidation of FGE is Cu²⁺. One example of an oxidationreagent that includes a reagent capable of oxidizing FGE and a reagentthat catalyzes the oxidation of FGE is a reagent that includes elementaloxygen as a terminal oxidant and a transition metal, such as, but notlimited to, Cu²⁺. These and other suitable oxidation reagents aredescribed below.

Activated FGE may be produced as part of an in vivo protein synthesismethod in which the FGE is expressed and activated within a host cell,which method may optionally include co-expression of a protein ofinterest containing an FGE recognition site in the host cell. That is,the FGE may be present in a host cell, and the FGE is treated to producean activated FGE by providing an oxidation reagent (e.g., Cu²⁺) in acell culture medium in which the host cell is being cultured. Forexample, the present inventors have found that treatment ofFGE-expressing cells with an oxidation reagent such as Cu²⁺ (byproviding Cu²⁺ in the cell culture medium) provides for FGE activationsuch that cysteine residues within FGE recognition sites of targetproteins co-expressed in the treated cells are converted toformylglycine residues at an increased yield as compared to conversionin cells not treated with Cu²⁺ but otherwise under identical conditions(see the Examples section below). For example, treatment ofFGE-expressing cells with an oxidation reagent as described herein mayproduce an increase in the population of activated FGE produced by theFGE-expressing cells. Similarly, treatment of FGE-expressing cells withan oxidation reagent as described herein may produce a decrease in thepopulation of FGE that is not activated FGE produced by theFGE-expressing cells.

Accordingly, in certain embodiments, the oxidation reagent is Cu²⁺, andtreating an FGE includes culturing a cell that includes a nucleic acidencoding the FGE in a cell culture medium that includes Cu²⁺, where theculturing is under conditions in which the FGE is expressed in the cell.In certain aspects, the present disclosure provides methods that includeculturing a cell that includes a nucleic acid encoding an FGE in a cellculture medium that includes Cu²⁺, where the culturing is underconditions in which the FGE is expressed in the cell.

The source of Cu²⁺ (e.g., a copper salt or other suitable Cu²⁺ source)and the Cu²⁺ concentration in the cell culture medium may be selected soas to be cell culture compatible, e.g., without affecting orsubstantially affecting cell viability, protein expression levels, etc.For example, when the host cell is a prokaryotic cell, the Cu²⁺ sourceand concentration may be selected so as to be compatible withprokaryotic cell culture. Similarly, when the host cell is, e.g., amammalian cell, the Cu²⁺ source and concentration may be selected so asto be compatible with mammalian cell culture. Examples of cultureconditions suitable for in vivo FGE activation in prokaryotic andmammalian cells are provided in the Examples section below.

In certain aspects, the Cu²⁺ is provided by addition of a copper salt tothe cell culture medium. Suitable copper salts include, but are notlimited to, copper sulfate (i.e., copper(II) sulfate, CuSO₄), coppercitrate, copper tartrate, copper nitrate, and any combination thereof.

The Cu²⁺ is present in the cell culture medium at a concentrationsuitable for FGE activation. In certain aspects, the Cu²⁺ is present inthe cell culture medium at a concentration of from 1 nM to 100 mM, suchas from 0.1 μM to 10 mM, from 0.5 μM to 5 mM, from 1 μM to 1 mM, from 2μM to 500 μM, from 3 μM to 250 μM, from 4 μM to 150 μM, or from 5 μM to100 μM (e.g., from 5 μM to 50 μM).

According to certain embodiments, the Cu²⁺ is present in the cellculture medium at a concentration of 1 nM or more, 10 nM or more, 100 nMor more, 1 μM or more, 5 μM or more, 10 μM or more, 20 μM or more, 30 μMor more, 40 μM or more, 50 μM or more, 100 μM or more, 200 μM or more,300 μM or more, 400 μM or more, 500 μM or more, 1 mM or more, 10 mM ormore, or 100 mM or more. In certain aspects, the Cu²⁺ is present in thecell culture medium at a concentration of 100 mM or less, 10 mM or less,1 mM or less, 500 μM or less, 400 μM or less, 300 μM or less, 200 μM orless, 100 μM or less, 50 μM or less, 40 μM or less, 30 μM or less, 20 μMor less, 10 μM or less, 5 μM or less, 1 μM or less, 100 nM or less, 10nM or less, or 1 nM or less.

Host cells suitable for in vivo FGE activation include, e.g.,prokaryotic cells (e.g., E. coli cells) and eukaryotic cells (e.g.,yeast cells, insect cells, mammalian cells, etc.). The cell may begenetically modified to express and FGE or interest and/or the FGE maybe endogenous to the cell.

Escherichia coli is an example of a prokaryotic host cell which may beused to practice the methods of producing an activated FGE. Othermicrobial hosts suitable for use include bacilli, such as Bacillussubtilis, and other enterobacteriaceae, such as Salmonella, Serratia,and various Pseudomonas species. In these prokaryotic hosts, one canmake expression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences and the like, for initiating and completingtranscription and translation.

Other microbes, such as yeast, are also useful for practicing themethods of producing an activated FGE. Saccharomyces (e.g., S.cerevisiae) and Pichia are examples of suitable yeast host cells, withsuitable vectors having expression control sequences (e.g., promoters),an origin of replication, termination sequences and the like as desired.Typical promoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilization.

In addition to microorganisms, mammalian cells may be employed topractice the methods of producing an activated FGE. The mammalian cellmay be a rodent cell, a human cell, or any other mammalian cell ofinterest, including but not limited to a cell selected from: a CHO cell,a HEK cell, a BHK cell, a COS cell, a Vero cell, a Hela cell, an NIH 3T3cell, a Huh-7 cell, a PC12 cell, a RAT1 cell, a mouse L cell, an HLHepG2cell, an NSO cell, a C127 cell, a hybridoma cell, a PerC6 cell, a CAPcell, and a Sp-2/0 cell. Expression vectors for these cells can includeexpression control sequences, such as an origin of replication, apromoter, and an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)),and necessary processing information sites, such as ribosome bindingsites, RNA splice sites, polyadenylation sites, and transcriptionalterminator sequences. Examples of suitable expression control sequencesare promoters derived from immunoglobulin genes, SV40, adenovirus,bovine papilloma virus, cytomegalovirus and the like. See Co et al., J.Immunol. 148:1149 (1992).

Culture conditions suitable for expressing FGEs in host cells aredescribed, for example in U.S. Pat. No. 7,985,783 and U.S. PatentApplication Publication No. US2011/0117621, the disclosures of which areincorporated herein by reference in their entireties for all purposes.The FGE may be endogenous to the host cell, or the host cell may berecombinant for a suitable FGE that is heterologous to the host cell.FGE expression can be provided by an expression system endogenous to theFGE gene (e.g., expression is provided by a promoter and other controlelements present in the native FGE gene of the host cell), or can beprovided by from a recombinant expression system in which the FGE codingsequence is operably linked to a heterologous promoter to provide forconstitutive or inducible expression. Use of a strong promoter toprovide high levels of FGE expression may be of particular interest incertain embodiments. The type of cell culture medium and componentstherein may be selected so as to be compatible with the particular celltype in which the FGE is expressed.

The methods may further include purifying the activated FGE from thecell and/or the oxidation reagent. Any convenient protein purificationprocedures may be used to isolate the activated FGE. See, e.g., Guide toProtein Purification, 2nd Edition (Burgess & Deutscher ed.) (AcademicPress, 2009) (ISBN: 9780123745361). For example, a lysate may beprepared from a cell that produces an activated FGE, and purified usingHPLC, size exclusion chromatography, gel electrophoresis, affinitychromatography, and the like.

Activated FGE may also be produced as part of an in vitro, cell-free,protein synthesis method, which method may optionally includeco-expression of a target polypeptide containing an FGE recognitionsequence in the reaction mixture. Accordingly, in certain aspects, themethods of the present disclosure include expressing the FGE in acell-free reaction mixture with an oxidation reagent, where theoxidation reagent can be Cu²⁺. According to these aspects, the cell-freereaction mixture is such that the FGE is not expressed in a cell, butrather in a reaction mixture suitable for in vitro protein synthesis.Exemplary cell-free reaction mixtures include, but are not limited to,cell-free extracts, cell lysates, and reconstituted translation systems,along with the nucleic acid template(s) for synthesis of an FGE and anyother desired proteins (e.g., a protein having an FGE recognition siteas described elsewhere herein).

The cell-free reaction mixture may include monomers for a macromoleculeto be synthesized, e.g. amino acids, nucleotides, etc., and suchco-factors, enzymes and any other necessary reagents, e.g. ribosomes,tRNA, polymerases, transcriptional factors, etc. In addition to theabove components such as a cell-free extract, nucleic acid template, andamino acids, materials specifically required for protein synthesis maybe added to the reaction. The materials may include salts, folinic acid,cyclic AMP, inhibitors for protein or nucleic acid degrading enzymes,inhibitors or regulators of protein synthesis, adjusters ofoxidation/reduction potentials, non-denaturing surfactants, buffercomponents, spermine, spermidine, putrescine, etc. Various cell-freesynthesis reaction systems are described, for example, in Kim, D. M. andSwartz, J. R. Biotechnol. Bioeng. 66: 180-8 (1999); Kim, D. M. andSwartz, J. R. Biotechnol. Prog. 16:385-90 (2000); Kim, D. M. and Swartz,J. R. Biotechnol. Bioeng. 74:309-16 (2001); Swartz et al, Methods MoLBiol. 267: 169-82 (2004); Kim, D. M. and Swartz, J. R. Biotechnol.Bioeng. 85: 122-29 (2004); Jewett, M. C. and Swartz, J. R., Biotechnol.Bioeng. 86: 19-26 (2004); Yin, G. and Swartz, J. R., Biotechnol. Bioeng.86: 188-95 (2004); Jewett, M. C. and Swartz, J. R., Biotechnol. Bioeng.87:465-72 (2004); Voloshin, A. M. and Swartz, J. R., Biotechnol. Bioeng.91:516-21 (2005). Additional conditions for the cell-free synthesis ofproteins of interest are described in International Publication No.WO2010/081110, the disclosure of which is incorporated herein byreference in its entirety.

In certain embodiments, cell-free protein synthesis offers certainadvantages over conventional in vivo protein expression methods.Cell-free systems can direct most, if not all, of the metabolicresources of the cell towards the exclusive production of one protein.Moreover, the lack of a cell wall and membrane components in vitro maybe advantageous since it allows for control of the synthesisenvironment. For example, tRNA levels can be changed to reflect thecodon usage of genes being expressed. The type or concentration of theoxidation reagent, the redox potential, pH, or ionic strength can alsobe altered with greater flexibility than with in vivo protein synthesisbecause concerns of cell growth or viability do not exist. Furthermore,direct recovery of purified, properly folded protein products can beeasily achieved.

Activated FGE may also be produced as part of an in vitro activationmethod. According to such embodiments, the FGE is not expressed in thepresence of an oxidation reagent. Such methods are based on the presentinventors' discovery that expressing an FGE (e.g., in a cell or acell-free translation system), purifying the FGE, and then treating theFGE with an oxidation reagent in vitro results in activation of the FGE.Such in vitro activated FGEs convert cysteine or serine residues withinFGE recognition sites of target proteins at an increased efficiency ascompared to conversion by non-activated FGEs under otherwise identicalconditions (see the Examples section below).

The oxidation reagent used for the activation of FGE may be selectedfrom any convenient oxidation reagent that is compatible with thedesired reaction conditions. For example, the oxidation reagent may beelemental oxygen, such as elemental oxygen as a terminal oxidant. Insome cases, elemental oxygen as a terminal oxidant may be provided asoxygen, a mixture of oxygen and hydrogen sulfide, oxygen under basicconditions, and the like.

In certain embodiments, the oxidation reagent is elemental oxygen, suchas elemental oxygen as a terminal oxidant, where the reaction iscatalyzed by a transition metal. In these instances, the oxidationreagent may include, but is not limited to, copper(II) (i.e., Cu²⁺) or asource of copper(II) (e.g., copper sulfate (CuSO₄), copper citrate,copper tartrate, copper nitrate, Fehling's reagent, Benedict's reagent,etc.), which can catalyze oxygen activation. In some instances, theoxidation reagent (e.g., Cu²⁺) that can catalyze oxygen activation isprovided in the presence of oxygen. For example, the oxidation reagentmay include oxygen and Cu²⁺, a source of Cu²⁺, or combinations thereof.

In certain embodiments, the oxidation reagent is copper(II) (i.e., Cu²⁺)or a source of copper(II) (i.e., a source of Cu²⁺). In some instances,the copper(II) or the source of copper(II) is provided in the presenceof oxygen. In some cases, the oxidation reagent is copper(II), such ascopper(II) and oxygen. In some cases, the oxidation reagent is a sourceof copper(II), such as a source of copper(II) and oxygen. In certaincases, the source of copper(II) is, but not limited to, copper sulfate,copper citrate, copper tartrate, Fehling's reagent, Benedict's reagent,combinations thereof, and the like. In some instances, the source ofcopper(II) is copper sulfate (CuSO₄).

Reaction conditions for the in vitro oxidation of proteins may varyaccording to the specific oxidation reagent employed. Example reactionconditions for the in vitro activation of FGE using example oxidationreagents are provided below in the Examples section.

Activation of the FGE may include combining in an FGE activationreaction mixture, an FGE, and an oxidation reagent. In some instances,the FGE activation reaction mixture is an aqueous solution. In certaincases, the FGE activation reaction mixture is a buffered aqueoussolution, e.g., an aqueous solution that includes a buffer, such as, butnot limited to, triethanolamine. In certain instances, the bufferedaqueous solution has a pH range compatible with FGE, such as a pH rangefrom 5 to 10, or from 5 to 9, or from 6 to 8, e.g., a pH of about 7,such as a pH of 7.4.

In certain embodiments, activation of the FGE may include combining inan FGE activation reaction mixture an FGE and an oxidation reagent,where the FGE is present in the FGE activation reaction mixture and theoxidation reagent is then added to the FGE activation reaction mixture.In other embodiments, activation of the FGE may include combining in anFGE activation reaction mixture, an FGE, and an oxidation reagent, wherethe oxidation reagent is present in the FGE activation reaction mixtureand the FGE is then added to the FGE activation reaction mixture.

In certain embodiments, the mol ratio of FGE to oxidation reagent in theFGE activation reaction mixture is 5:1, or 4:1, or 3:1, or 2:1, or 1:1,or 1:2, or 1:3, or 1:4, or 1:5. In some instances, the mol ratio of FGEto oxidation reagent in the FGE activation reaction mixture is 1:2.

In certain cases, after the FGE and the oxidation reagent are combinedin the FGE activation reaction mixture, the FGE activation reactionmixture is mixed. Any convenient method for mixing the FGE activationreaction mixture may be used, such as stirring, vortexing, and the like.The FGE activation reaction mixture may be mixed for a period of time toallow sufficient activation of the FGE to activated FGE, such as 15 minor more, or 30 min or more, or 45 min or more, or 1 hr or more, or 2 hror more, or 3 hr or more, or 4 hr or more, or 5 hr or more. In somecases, the FGE activation reaction mixture is mixed for 1 hr. Mixing ofthe FGE activation reaction mixture may be performed at about roomtemperature, e.g., a temperature ranging from 20° C. to 30° C., such as25° C. Temperatures suitable for activation may vary according to theparticular oxidation reagent employed.

Sufficient activation of the FGE to activated FGE may be measured usingan FGE activity assay as described herein. After sufficient activationof the FGE to activated FGE, the activated FGE may be separated from theFGE activation reaction mixture. Any convenient method for proteinseparation may be used to separate the activated FGE from the FGEactivation reaction mixture, such as, but not limited to, dialysis,buffer exchange, diafiltration, precipitation, ion exchangechromatography, affinity chromatography, electrophoresis, and the like.In some instances, the activated FGE may be separated from the FGEactivation reaction mixture by buffer exchange.

Also provided by the present disclosure is an activated FGE produced byany of the methods described herein for producing an activated FGE.According to certain embodiments, the activated FGE is not purified fromthe oxidation reagent. For example, the FGE may be present in the cellor reaction mixture in which the FGE was treated with the oxidationreagent. In other aspects, the activated FGE is separated from the FGEactivation reaction mixture using a suitable protein separationprocedure, including any such separation procedures described elsewhereherein.

Methods of Using Activated FGEs

The activated FGEs of the present disclosure find use in a variety of invivo and in vitro applications in which it is desirable, e.g., toconvert a cysteine residue or a serine residue of an FGE recognitionsite in a protein of interest (or “target protein”) to a formylglycineresidue. The aldehyde group of the formylglycine residue is useful,e.g., for site-specifically conjugating an agent of interest (e.g., atherapeutic agent, an imaging agent, etc.) to the target protein.Accordingly, the present disclosure provides methods of using activatedFGEs.

In certain aspects, provided are methods of producing a protein thatincludes a formylglycine residue. The methods include combining anactivated formylglycine-generating enzyme (FGE) with a protein thatincludes an FGE recognition site, under conditions in which theactivated FGE converts a cysteine residue or a serine residue of the FGErecognition site to a formylglycine residue, to produce a proteinincluding a formylglycine residue.

In some embodiments, the activated FGE and the protein having the FGErecognition site are combined in a reaction mixture that includes areducing agent. In some embodiments, the reducing agent promotesconversion of the cysteine residue or the serine residue of the FGErecognition site to the formylglycine residue. In some embodiments, thereducing agent is 2-mercaptoethanol. As used herein, promoting theconversion of the cysteine residue or the serine residue of the FGErecognition site to the formylglycine residue refers to an increase inthe amount (e.g., concentration) of fGly produced by the activated FGEas compared to a conversion reaction in the absence of the reducingagent. In certain cases, the reducing agent increases the amount of fGlyproduced by the activated FGE by 5% or more, or 10% or more, or 15% ormore, or 20% or more, or 25% or more, or 30% or more, or 35% or more, or40% or more, or 45% or more, or 50% or more, or 55% or more, or 60% ormore, or 65% or more, or 70% or more, or 75% or more, or 80% or more, or85% or more, or 90% or more, or 95% or more, or 100% or more, ascompared to a conversion reaction in the absence of the reducing agent.In certain embodiments, the reducing agent increases the amount of fGlyproduced by the activated FGE by 50% or more, as compared to aconversion reaction in the absence of the reducing agent.

In Vivo Conversion

The protein that includes a formylglycine residue may be produced withina host cell as part of an in vivo protein synthesis and conversionmethod. According to certain embodiments, combining an activated FGEwith a protein including an FGE recognition site includes culturing acell that includes an FGE and a protein including an FGE recognitionsite. The culturing is in a cell culture medium that includes Cu²⁺ asthe oxidation reagent, under cell culture conditions in which the FGEconverts a cysteine residue or a serine residue of the FGE recognitionsite to a formylglycine residue. In certain aspects, the cell includesnucleic acids that encode the FGE and the protein including an FGErecognition site, such that the FGE and protein including an FGErecognition site are co-expressed in the cell.

The source of Cu²⁺ (e.g., a copper salt or other suitable Cu²⁺ source)and the Cu²⁺ concentration in the cell culture medium may be selected soas to be cell culture compatible, e.g., without affecting orsubstantially affecting cell viability, protein expression levels, etc.For example, when the host cell is a prokaryotic cell, the Cu²⁺ sourceand concentration may be selected so as to be compatible withprokaryotic cell culture. Similarly, when the host cell is, e.g., amammalian cell, the Cu²⁺ source and concentration may be selected so asto be compatible with mammalian cell culture. Examples of cultureconditions suitable for in vivo FGE activation in prokaryotic andmammalian cells are provided in the Examples section below.

In certain aspects, the Cu²⁺ is provided by addition of a copper salt tothe cell culture medium. Suitable copper salts include, but are notlimited to, copper sulfate (i.e., copper(II) sulfate, CuSO₄), coppercitrate, copper tartrate, copper nitrate, and any combination thereof.

The Cu²⁺ is present in the cell culture medium at a concentrationsuitable for FGE activation. In certain aspects, the Cu²⁺ is present inthe cell culture medium at a concentration of from 1 nM to 100 mM, suchas from 0.1 μM to 10 mM, from 0.5 μM to 5 mM, from 1 μM to 1 mM, from 2μM to 500 μM, from 3 μM to 250 μM, from 4 μM to 150 μM, or from 5 μM to100 μM (e.g., from 5 μM to 50 μM).

According to certain embodiments, the Cu²⁺ is present in the cellculture medium at a concentration of 1 nM or more, 10 nM or more, 100 nMor more, 1 μM or more, 5 μM or more, 10 μM or more, 20 μM or more, 30 μMor more, 40 μM or more, 50 μM or more, 100 μM or more, 200 μM or more,300 μM or more, 400 μM or more, 500 μM or more, 1 mM or more, 10 mM ormore, or 100 mM or more. In certain aspects, the Cu²⁺ is present in thecell culture medium at a concentration of 100 mM or less, 10 mM or less,1 mM or less, 500 μM or less, 400 μM or less, 300 μM or less, 200 μM orless, 100 μM or less, 50 μM or less, 40 μM or less, 30 μM or less, 20 μMor less, 10 μM or less, 5 μM or less, 1 μM or less, 100 nM or less, 10nM or less, or 1 nM or less.

Host cells suitable for co-expression of an FGE and a protein includingan FGE recognition site include, e.g., prokaryotic cells (e.g., E. colicells) and eukaryotic cells (e.g., yeast cells, insect cells, mammaliancells, etc.).

Escherichia coli is an example of a prokaryotic host cell which may beused to practice the methods of producing a protein that includes aformylglycine residue. Other microbial hosts suitable for use includebacilli, such as Bacillus subtilis, and other enterobacteriaceae, suchas Salmonella, Serratia, and various Pseudomonas species. In theseprokaryotic hosts, one can make expression vectors, which will typicallycontain expression control sequences compatible with the host cell(e.g., an origin of replication). In addition, any number of a varietyof well-known promoters will be present, such as the lactose promotersystem, a tryptophan (trp) promoter system, a beta-lactamase promotersystem, or a promoter system from phage lambda. The promoters willtypically control expression, optionally with an operator sequence, andhave ribosome binding site sequences and the like, for initiating andcompleting transcription and translation.

Other microbes, such as yeast, are also useful for practicing themethods of producing a protein that includes a formylglycine residue.Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitableyeast host cells, with suitable vectors having expression controlsequences (e.g., promoters), an origin of replication, terminationsequences and the like as desired. Typical promoters include3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeastpromoters include, among others, promoters from alcohol dehydrogenase,isocytochrome C, and enzymes responsible for maltose and galactoseutilization.

In addition to microorganisms, mammalian cells may be employed topractice the methods of producing a protein that includes aformylglycine residue. The mammalian cell may be a rodent cell, a humancell, or any other mammalian cell of interest, including but not limitedto a cell selected from: a CHO cell, a HEK cell, a BHK cell, a COS cell,a Vero cell, a Hela cell, an NIH 3T3 cell, a Huh-7 cell, a PC12 cell, aRAT1 cell, a mouse L cell, an HLHepG2 cell, an NSO cell, a C127 cell, ahybridoma cell, a PerC6 cell, a CAP cell, and a Sp-2/0 cell. Expressionvectors for these cells can include expression control sequences, suchas an origin of replication, a promoter, and an enhancer (Queen et al.,Immunol. Rev. 89:49 (1986)), and necessary processing information sites,such as ribosome binding sites, RNA splice sites, polyadenylation sites,and transcriptional terminator sequences. Examples of suitableexpression control sequences are promoters derived from immunoglobulingenes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus and thelike. See Co et al., J. Immunol. 148:1149 (1992).

Cell-Free Conversion in the Context of an In Vitro Transcription Method

The protein that includes a formylglycine residue may be produced aspart of an in vitro, cell-free, protein synthesis and conversion method.In certain aspects, the methods include expressing an FGE and theprotein including an FGE recognition site in a cell-free reactionmixture that includes an oxidation reagent (e.g., Cu²⁺) under conditionsin which the FGE converts a cysteine residue or a serine residue of theFGE recognition site to a formylglycine residue. According to theseaspects, the cell-free reaction mixture is such that the FGE and theprotein including an FGE recognition site are not expressed in a cell,but rather in a reaction mixture suitable for in vitro proteinsynthesis. Exemplary cell-free reaction mixtures include, but are notlimited to, cell-free extracts, cell lysates, and reconstitutedtranslation systems, along with nucleic acid templates for synthesis ofan FGE and the protein including an FGE recognition site. The componentsof the cell free reaction mixture may be as described hereinabove withrespect to the in vitro, cell-free, protein synthesis methods forproducing an activated FGE.

In Vitro Conversion Using Activated FGE

The protein that includes a formylglycine residue may be produced aspart of an in vitro conversion method, in which an activated FGE and aprotein including an FGE recognition site are combined in a cell-freereaction mixture. According to these embodiments, the FGE and theprotein including an FGE recognition site are not expressed in thecell-free reaction mixture. Such methods are based on expressing an FGE(e.g., in a cell or a cell-free translation system), purifying the FGE,and then treating the FGE with an oxidation reagent in vitro to produceactivation of the FGE. Such in vitro activated FGEs convert cysteine orserine residues within FGE recognition sites of proteins that includeFGE recognition sites at an increased efficiency as compared toconversion by non-activated FGEs under otherwise identical conditions(see the Examples section below).

In some embodiments, the reaction mixture includes a copper(II) ion(i.e., Cu²⁺), or a source of copper(II) ions, e.g., copper sulfate(CuSO₄), copper citrate, copper tartrate, and the like. Expressing theFGE and the protein having the FGE recognition site in the cell-freereaction mixture containing Cu²⁺ may occur under conditions in which theFGE converts a cysteine residue or a serine residue of the FGErecognition site to a formylglycine (Fgly) residue, as described herein.

In certain embodiments, the in vitro method of producing a proteinhaving an FGly residue includes combining an activated FGE and theprotein having the FGE recognition site in a cell-free reaction mixture.In these embodiments, the FGE may be activated to form an activated FGEprior to combining the activated FGE and the protein having the FGErecognition site. By “activated” is meant that the activated FGE has agreater activity as compared to an FGE that has not been activated,e.g., a greater activity for the conversion of a cysteine or a serineresidue in of the FGE recognition site to an FGly. For instance, anactivated FGE may have an activity (as measured using an FGE activityassay as described herein) that is 1.5 times or more, 2 times or more, 5times or more, 10 times or more, 20 times or more, 30 times or more, 40times or more, 50 times or more, 60 times or more, 70 times or more, 80times or more, 90 times or more, 100 times or more active, such as 200times or more, including 300 times or more, or 400 times or more, or 500times or more, or 600 times or more, or 700 times or more, or 800 timesor more, or 900 times or more, or 1000 times or more, or 1500 times ormore, or 2000 times or more, or 2500 times or more, or 3000 times ormore, or 3500 times or more, or 4000 times or more, or 4500 times ormore, or 5000 times or more, or 5500 times or more, or 6000 times ormore, or 6500 times or more, or 7000 times or more, or 7500 times ormore, or 8000 times or more, or 8500 times or more, or 9000 times ormore, or 9500 times or more, or 10,000 times or more active than an FGEthat has not been activated. In certain instances, the activated FGE hasan activity (as measured using an FGE activity assay as describedherein) that is 3000 times or more active than an FGE that has not beenactivated. In some cases, the activated FGE has an activity (as measuredusing an FGE activity assay as described herein) that is 1.5 to 10,000times more, 2 to 10,000 times more, 5 to 10,000 times more, 10 to 10,000times more, 20 to 10,000 times more, 30 to 10,000 times more, 40 to10,000 times more, 50 to 10,000 times more, 60 to 10,000 times more, 70to 10,000 times more, 80 to 10,000 times more, 90 to 10,000 times more,100 to 10,000 times more active, such as 500 to 9000 times more,including 1000 to 8000 times more, or 1000 to 7000 times more, or 1000to 6000 times more, or 1000 to 5000 times more, or 1000 to 4000 times,or 2000 to 4000 times more active than an FGE that has not beenactivated. In certain instances, the activated FGE has an activity (asmeasured using an FGE activity assay as described herein) that is 2000to 4000 times more active than an FGE that has not been activated.

In certain embodiments, the FGE may be activated to form an activatedFGE prior to combining the activated FGE and the protein having the FGErecognition site. As such, in some instances, the method includesactivating the FGE with an oxidation reagent prior to combining theactivated FGE with the protein having the FGE recognition site.

The oxidation reagent used for the activation of FGE may be selectedfrom any convenient oxidation reagent that is compatible with thedesired reaction conditions. For example, the oxidation reagent may beelemental oxygen, such as elemental oxygen as a terminal oxidant. Insome cases, elemental oxygen as a terminal oxidant may be provided asoxygen, a mixture of oxygen and hydrogen sulfide, oxygen under basicconditions, and the like.

In certain embodiments, the oxidation reagent is elemental oxygen, suchas elemental oxygen as a terminal oxidant, where the reaction iscatalyzed by a transition metal. In these instances, the oxidationreagent may include, but is not limited to, copper(II), a source ofcopper(II) (e.g., copper sulfate, copper citrate, copper tartrate,Fehling's reagent, Benedict's reagent, etc.), which can catalyze oxygenactivation. In some instances, the oxidation reagent (e.g., Cu²⁺) thatcan catalyze oxygen activation is provided in the presence of oxygen.For example, the oxidation reagent may include oxygen and Cu²⁺, a sourceof Cu²⁺, or combinations thereof.

In certain embodiments, the oxidation reagent is copper(II) or a sourceof copper(II). In some instances, the copper(II) or the source ofcopper(II) is provided in the presence of oxygen. In some cases, theoxidation reagent is copper(II), such as copper(II) and oxygen. In somecases, the oxidation reagent is a source of copper(II), such as a sourceof copper(II) and oxygen. In certain cases, the source of copper(II) is,but not limited to, copper sulfate, copper citrate, copper tartrate,Fehling's reagent, Benedict's reagent, combinations thereof, and thelike. In some instances, the source of copper(II) is copper sulfate.

As described above, in certain embodiments, the mol ratio of FGE tooxidation reagent is 5:1, or 4:1, or 3:1, or 2:1, or 1:1, or 1:2, or1:3, or 1:4, or 1:5. In some instances, the mol ratio of FGE tooxidation reagent in the FGE activation reaction mixture is 1:2.

As described above, in certain embodiments, the in vitro method ofproducing a protein having an FGly residue includes combining anactivated FGE and the protein having the FGE recognition site in acell-free reaction mixture. As described above, the FGE may be activatedto form an activated FGE prior to combining the activated FGE and theprotein having the FGE recognition site. Approaches for producing anactivated FGE are described in detail above in the description of themethods for producing activated FGEs.

In certain embodiments, the ratio of the activated FGE to the proteinhaving the FGE recognition site (i.e., the ratio of activated FGE toprotein in the conversion reaction) is 200% by mol or less, such as 150%by mol or less, or 100% by mol or less, or 75% by mol or less, or 50% bymol or less, or 25% by mol or less, or 10% by mol or less, or 5% by molor less, or 3% by mol or less, or 1% by mol or less, or 0.7% by mol orless, or 0.5% by mol or less, or 0.4% by mol or less, or 0.3% by mol orless, or 0.2% by mol or less, or 0.1% by mol or less, or 0.07% by mol orless, or 0.05% by mol or less, or 0.03% by mol or less, or 0.01% by molor less, or 0.005% by mol or less. In certain embodiments, the ratio ofthe activated FGE to the protein having the FGE recognition site is 0.1%by mol or less, such as 0.1% by mol.

According to certain embodiments, the methods of producing a proteincomprising a formylglycine residue further include conjugating an agentto the produced protein via the aldehyde moiety of the formylglycineresidue. In certain aspects, the agent is a therapeutic agent, animaging agent, an agent that increases serum half-life of the proteinupon administration to a subject, an agent that reduces immunogenicityof the protein upon administration to a subject, an agent that increasesimmunogenicity of the protein upon administration to a subject (e.g.,when the protein is a vaccine), or any other desirable agents forconjugation to the produced protein.

According to certain embodiments, the methods include conjugating atherapeutic agent to the protein. In certain aspects, the therapeuticagent is selected from a cytotoxic agent, an antiproliferative agent, anantineoplastic agent, an antibiotic agent, an antifungal agent, and anantiviral agent.

According to certain embodiments, the agent of interest is an imagingagent. The imaging agent may be, e.g., a fluorescent dye, anear-infrared (NIR) imaging agent, and a single-photon emission computedtomography (SPECT)/CT imaging agent, a nuclear magnetic resonance (NMR)imaging agent, a magnetic resonance imaging (MRI) agent, apositron-emission tomography (PET) agent, an x-ray imaging agent, acomputed tomography (CT) imaging agent, a K-edge imaging agent, anultrasound imaging agent, a photoacoustic imaging agent, an acousticoptical imaging agent, microwave imaging agent, a nuclear imaging agent,and combinations thereof.

The agent of interest is provided as a component of a reactive partnerfor reaction with an aldehyde of the formylglycine residue of theproduced protein. A wide range of commercially available reagents can beused to attach an agent of interest to the formylglycine residue of theprotein. For example, aminooxy, hydrazide, or thiosemicarbazidederivatives of a number of agents of interest are suitable reactivepartners, and are readily available or can be generated using standardchemical methods.

Compositions

The present disclosure provides compositions, which in certain aspectsare compositions useful in practicing the methods of the presentdisclosure.

In a first aspect, provided is a composition that includes a cellculture medium that includes an oxidation reagent compatible with cellculture, such as Cu²⁺ and a cell present in the cell culture medium,where the cell express an FGE.

The Cu²⁺ may be present in the cell culture medium at any desiredconcentration, including concentrations suitable for FGE activation. Incertain aspects, the Cu²⁺ is present in the cell culture medium at aconcentration of from 1 nM to 100 mM, such as from 0.1 μM to 10 mM, from0.5 μM to 5 mM, from 1 μM to 1 mM, from 2 μM to 500 μM, from 3 μM to 250μM, from 4 μM to 150 μM, or from 5 μM to 100 μM (e.g., from 5 μM to 50μM).

According to certain embodiments, the Cu²⁺ is present in the cellculture medium at a concentration of 1 nM or more, 10 nM or more, 100 nMor more, 1 μM or more, 5 μM or more, 10 μM or more, 20 μM or more, 30 μMor more, 40 μM or more, 50 μM or more, 100 μM or more, 200 μM or more,300 μM or more, 400 μM or more, 500 μM or more, 1 mM or more, 10 mM ormore, or 100 mM or more. In certain aspects, the Cu²⁺ is present in thecell culture medium at a concentration of 100 mM or less, 10 mM or less,1 mM or less, 500 μM or less, 400 μM or less, 300 μM or less, 200 μM orless, 100 μM or less, 50 μM or less, 40 μM or less, 30 μM or less, 20 μMor less, 10 μM or less, 5 μM or less, 1 μM or less, 100 nM or less, 10nM or less, or 1 nM or less.

The cell present in the cell culture medium may be a prokaryotic cell(e.g., an E. coli cell) a eukaryotic cell (e.g., a yeast cell, an insectcell, a mammalian cell, etc.). The cell present in the cell culturemedium may include a nucleic acid template from which the FGE isexpressed, and optionally, a nucleic acid template for expression of aprotein that includes an FGE recognition site.

In certain aspects, the cell present in the cell culture medium is amammalian cell selected from a CHO cell, a HEK cell, a BHK cell, a COScell, a Vero cell, a Hela cell, an NIH 3T3 cell, a Huh-7 cell, a PC12cell, a RAT1 cell, a mouse L cell, an HLHepG2 cell, an NSO cell, a C127cell, a hybridoma cell, a PerC6 cell, a CAP cell, and a Sp-2/0 cell.

Also provided are cell-free compositions. In certain aspects, thecell-free compositions include an activated FGE and a buffer. Forexample, the activated FGE may be present in a buffer. Any activated FGEof interest may be included in the cell-free composition, including anactivated FGE produced by any of the methods described herein forproducing an activated FGE. According to certain embodiments, theactivated FGE is a purified activated FGE which has been separated froma reaction mixture (e.g., an in vivo or cell-free protein synthesisreaction mixture, and FGE activation reaction mixture, etc.), e.g., bydialysis, buffer exchange, diafiltration, precipitation, ion exchangechromatography, affinity chromatography, electrophoresis, and/or thelike.

A buffer suitable for maintaining the activated FGE in an activated formmay be employed. In certain aspects, the buffer is suitable forproviding a buffered aqueous solution having a pH range compatible withthe activated FGE, such as a pH range from 5 to 9, or from 6 to 8, e.g.,a pH of about 7, such as a pH of 7.4.

The cell-free compositions of the present disclosure may further includea protein that includes an FGE recognition site. Any protein of interesthaving an FGE recognition site may be present in the cell-freecomposition, including any of the proteins described herein above in thesection relating to proteins that include FGE recognition sites, e.g.,an antibody or antibody fragment of interest having an FGE recognitionsite, etc.

Kits

Aspects of the present disclosure further include kits. Kits accordingto certain embodiments of the present disclosure find use in practicingthe methods of the present disclosure.

According to one embodiment, provided is a kit that includes anactivated FGE, and instructions for using the activated FGE to convert acysteine residue or a serine residue present in an FGE recognition siteof a protein to a formylglycine residue. Such kits may further include abuffer for use in preparing a reaction mixture in which the activatedFGE may be used to convert a cysteine residue or a serine residuepresent in an FGE recognition site of a protein to a formylglycineresidue. The FGE and the buffer may be provided in the same or differentcontainers (e.g., tubes). A protein (e.g., an antibody or any otherprotein of interest) that includes a recognition site for the activatedFGE may also be included in the kit.

Also provided are kits that include a nucleic acid that encodes an FGE,and an oxidation reagent. In certain aspects, the nucleic acid thatencodes an FGE is an expression vector suitable for expressing the FGEin a prokaryotic or eukaryotic (e.g., mammalian) cell upontransformation or transfection of the vector into the cell. Theoxidation reagent may be any suitable oxidation reagent for activatingthe FGE, including any of the oxidation reagents described above in thesections relating to the methods of the present disclosure. In certainaspects, the oxidation reagent is Cu²⁺. The kit may further includecells (e.g., any of the prokaryotic or eukaryotic cells describedherein) suitable for expressing the FGE encoded by the nucleic acid.Instructions, e.g., for activating the FGE with the oxidation reagentand/or transforming or transfecting a cell type of interest with thenucleic acid, may be included in the kit.

Components of the kits of the present disclosure may be present inseparate containers, or multiple components may be present in a singlecontainer.

In addition to the above-mentioned components, a kit of the presentdisclosure may further include instructions for using the components ofthe kit, e.g., to practice the methods of the present disclosure. Theinstructions are generally recorded on a suitable recording medium. Forexample, the instructions may be printed on a substrate, such as paperor plastic, etc. As such, the instructions may be present in the kits asa package insert, in the labeling of the container of the kit orcomponents thereof (i.e., associated with the packaging or subpackaging)etc. In other embodiments, the instructions are present as an electronicstorage data file present on a suitable computer readable storagemedium, e.g., CD-ROM, Blu-Ray, Hard Disk Drive (HDD), computer readablememory (e.g., flash drive), etc. In yet other embodiments, the actualinstructions are not present in the kit, but means for obtaining theinstructions from a remote source, e.g. via the internet, are provided.An example of this embodiment is a kit that includes a web address wherethe instructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use embodiments of the present disclosure, and are not intendedto limit the scope of what the inventors regard as their invention norare they intended to represent that the experiments below are all or theonly experiments performed. Efforts have been made to ensure accuracywith respect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. By “average” is meant the arithmeticmean. Standard abbreviations may be used, e.g., bp, base pair(s); kb,kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h orhr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt,nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c.,subcutaneous(ly); and the like.

EXAMPLES

Equipment

Micropipettors (calibrated annually): ResearchPlus 2.5, 10, 20, 100,200, 1000 (Eppendorf); Serological Pipettor: Pipettor Plus (Omega);Balances (Calibrated monthly): XS4001S, NewClassic MF (ML204), AT200(Mettler Toledo); pH measurement (calibrated monthly): SevenCompact(Mettler Toledo), InLab Expert Pro electrode (Mettler Toledo), OrionStandards (Thermo Scientific); SDS-PAGE: PowerPac Basic (Bio-Rad),Mini-PROTEAN® Tetra System (Bio-Rad); PAGE gel imaging: Image Quant LAS4000 (GE Healthcare); Well plate washer: ELx405 (BioTek); Well platereader: SpectraMax M5 (Molecular Devices); Centrifuges: Sorvall RC 6Plus (Thermo Scientific), 5415 R & 5810 R (Eppendorf); Reaction mixer:Thermomixer R (Eppendorf), Vortexer: vortex genie 2 (ScientificIndustries); Shakers: Lab Companion SI-600R (JEIO Tech Co. Ltd.),Shaking Incubator (Shel Labs); PCR cycler: Mastercycler Pro (Eppendorf);sterilization: Hiclave (Hirayama).

Absorption Spectroscopy

UV and visible absorption spectra were recorded on a NanoDrop™ 2000(Thermo Scientific) spectrophotometer with manufacturer-suppliedsoftware, or on a GENESYS™ 10S spectrophotometer (Thermo Scientific)controlled with VISIONIite™ software. Spectrometers were calibrated withNIST-traceable potassium dichromate standards for photometric accuracy(Starna Scientific and Thermo Scientific).

Reversed-phase HPLC

Reversed-phase High Performance Liquid Chromatography was performed onan 1100/1200 series instrument (Agilent Technologies) controlled withAgilent OpenLAB CDS Chemstation Edition. The instrument included anin-line solvent degasser, analytical quaternary pump, vial auotsampler,thermostatted column compartment, and diode array detector.Chromatography was achieved on an Aeris™ core-shell 250×2.1 mm XB-C18Widepore column (Phenomenex, Inc.). Area under the curve (AUC) wascalculated with Chemstation (Agilent).

LC/MS

Mass Spectrometry data were collected on a 4000 QTRAP® mass spectrometer(AB Sciex) with an 1100 series HPLC (Agilent Technologies) that includedan inline solvent degasser, analytical binary pump, and a thermostattedvial/wellplate autosampler. Chromatography was performed on a Jupiter™150×1.0 mm C18 column (Phenomenex, Inc.) enclosed in a butterfly columnheater set to 65° C. with a PST-CHC controller (Phoenix S&T).Calculation of LC-MRM (multiple reaction monitoring)/MS transitionmasses and integration of the resulting data was performed with Skyline2.6.

FPLC

Fast Performance Liquid Chromatography was performed on a GE HealthcareAkta Protein Purification System consisting of the UPC-900, P-900 andFrac-950 modules. Nickel affinity chromatography was performed with aHisTrap® Excel 5 mL column (GE Healthcare). Gel filtration was performedby hand with disposable Sephadex® G-25 columns (GE Healthcare).

ICP-MS

Inductively-coupled plasmon mass spectrometry (ICP-MS) was performed bythe Catalent Center for Excellence in Analytical Services (Morrisville,N.C.). The ratio of copper and calcium were calculated as a mol ratiobased upon protein concentrations measured from 280 nm absorptionintensity of enzyme stock solutions (Table 3).

TABLE 3 FGE preparations contain both copper and calcium as measured byICP-MS. [Pro- Ca Cu Prepa- tein] Ca Ca (ra- Cu Cu (ra- ration (μM)*(μg/L) (μM) tio) (μg/L) (uM) tio) Sc-FGE 131.4 375 373.75 2.84 111 1.740.01 Hs-cFGE 204.3 98 97.4 0.48 4368 68.73 0.34 Sc-FGE + 64.5 55 54.990.85 4768 75.03 1.16 Cu Hs-FGE + 69.7 106 105.59 1.51 6112 96.18 1.38 Cu*Protein concentrations were measured using the absorption at 280 nm ofstock protein solutions. Extinction coefficients for protein werecalculated from the primary sequence of the enzyme using the analysistools on the ExPASy bioinformatics server. For Hs-cFGE, ε = 85,745M⁻¹cm⁻¹ and MW = 33,286 Da; for Sc-FGE, ε = 88,000 M⁻¹cm−1 and MW =37,432 Da.

Competitive Enyzme-Linked Immunosorbent Assay (ELISA) Assessment ofAntibody Affinity

ELISA plates (Maxisorp) were coated with 1 μg/mL of His-tagged CD22(Sino Biological 11958-H08H) at 1 μg/mL in PBS for 16 h. at 4° C. Theplate was then washed four times with PBS 0.1% Tween-20 (PBS-T), andthen blocked with 200 μL ELISA blocker (Thermo). In a second plate, an11-point standard curve was prepared from serial 3-fold dilutions (50μL+100 μL) of a stock solution of the competitor antibody at 20 μg/mLinto ELISA blocker. Next, biotin-tagged CD22 (100 μL of 10 ng/mL) wasadded at to all of the wells. Finally, the mixture of competitor andbiotin-tagged CD22 was transferred to the coated ELISA plate (100μL/well), which was incubated at RT for 1-2 h with shaking. The finalconcentration of biotin-tagged CD22 was 5 ng/mL and the highestconcentration of the competitor was 10 μg/mL. The plate was washed fourtimes with PBS-T; then, 100 μL of a 1:10,000 dilution (into blockingbuffer) of Streptavidin-HRP (Thermo 21130) was added and the plate wasincubated at RT with shaking for 1-2 h. Next, the plate was washed fourtimes with PBS-T, and developed with 1-Step Ultra TMB (100 μL/well,Pierce #34038). The reaction was quenched with 50 μl 2 M sulfuric acid,and absorbance was measured at 450 nm. EC₅₀ values were determined bynonlinear regression.

Estimation of Uncertainty

When displayed on bar graphs, measurements of uncertainty represent 95%confidence intervals calculated from the standard error of the mean fora sample size (n) of three or more using the following equation:

${error} = {1.96 \times \left( \frac{s}{\sqrt{n}} \right)}$

where s is the measured standard deviation for n samples. Errorsreported for the enzyme kinetic parameters represent the estimate oferror calculated from the minimized sum of squares found duringnonlinear regression of activity data to the Michaelis-Menten equation:

$y = \frac{E_{t} \cdot k_{cat} \cdot x}{\left( {K_{M} + x} \right)}$

where E_(t) is the total enzyme in solution, and k_(cat) and K_(M) arethe standard enzymatic parameters specified by the STRENDA commission.Units of enzyme specific activity are the katal, where 1 kat=1 mol·s⁻¹.Kinetic parameters were determined from nonlinear regression of[substrate] v. initial velocity using GraphPad prism.

Area under the curve (AUC) for HPLC runs was calculated by theChemstation software using the “new exponential” algorithm and settingslope sensitivity of 1 mAU. Kinetic parameters were determined fromnonlinear regression of [substrate] v. initial velocity using GraphPadPrism.

Software

All instrument data collection workstations were operated with PCsrunning Microsoft Windows OS and instrument control software as providedby the manufacturers. Data analysis was performed either on a PC runningMS windows or an Apple iMac using OS X 10.10. Linear and nonlinearregression was performed using Graph Pad Prism 6.0e. Integration of HPLCchromatograms was performed using OpenLAB CDS Chemstation Edition.Calculation of LC-MRM/MS transition masses and integration of theresulting data was performed with Skyline 2.6 (MacCoss Lab, Universityof Washington). Statistical calculations were performed with MicrosoftExcel. Graphs and figures were prepared with Adobe Creative Suite.Rendering of protein crystal structures was performed with open sourcePyMOL 1.7.

Materials

Reagents, Materials, Chemicals, and Abbreviations

All water used was deionized 18 MO) was from a Milli-Q Integral 5 system(EMD Millipore). Commercially available chemicals were reagent grade (orhigher, as indicated). Chemicals and materials were sourced as follows:Sigma-Aldrich—β-mercaptoethanol (βME), trifluoroacetic acid (TFA),methoxylamine hydrate, copper(II) sulfate pentahydrate, streptomycinsulfate, iodoacetamide (IAA); Acros—formic acid, triethanolamine (TEAM),dithiothreitol (DTT), glycerol, glucose, potassium phosphate monobasic,potassium phosphate dibasic; Fisher Scientific—imidazole, tyrptone,yeast extract, sodium chloride; Honeywell Burdick &Jackson—acetonitrile+0.1% TFA (HPLC grade), acetonitrile+0.1% formicacid (HPLC grade); Thermo Scientific—tris(carboxyethyl) phosphine,(TCEP), PageRuler Plus Protein Standard; Bio-Rad Laboratories—10×protein assay; IBI scientific—isopropylthiogalactoside (IPTG);Amresco—ampicillin sodium salt (USP); Roche diagnostics—cOmplete-miniEDTA-free protease inhibitor cocktail; EMD Millipore—BugBuster® MasterMix; Invitrogen—Ultracompetent E. coli BL21(DE3) cells.

Antibody Production

For antibody production, GPEx technology was used to generate bulkstable pools of antibody-expressing cells. Antibodies were purified fromthe conditioned medium using Protein A chromatography (MabSelect, GEHealthcare Life Sciences).

Peptide Synthesis

All peptide synthesis was performed on solid phase and purified to ≥95%.

Identification of Biological Materials

Sc-FGE—UniProt accession: Q9F3C7; PDB 2Q17.

Hs-FGE—UniProt accession: Q8NBK3; PDB 1Y1E.

Example 1: Cu²⁺ Increases the Conversion Efficiency of FGE in MammalianCells In Vivo

The effect on conversion in the presence of Cu²⁺ (copper sulfate) in thecell culture medium of mammalian cells expressing FGEs and proteins thatinclude FGE recognition sites was investigated. CHO cells expressing arecombinant human FGE and recombinant αHER2 mAb (heavy and light chainin same cell) containing the FGE recognition sequence LCTPSR werecultured FortiCHO media with the CellBoost4 feed, Efficient Feed C, andEfficient Feed C, supplemented with glucose and/or galactose. FortiCHOmedia (Life Technologies), CellBoost4 (Thermo Scientific) feed andEfficient Feed C (Life Technologies).

A variety of conditions were tested, and the results are summarizedbelow.

As shown in FIG. 1 (Panel A), Cu²⁺ (50 μM Day 0) increased conversion inFortiCHO media with the CellBoost4 feed, Efficient Feed C, and EfficientFeed C+glucose. As shown in FIG. 1 (Panel B), Cu²⁺ (50 μM Day 0)improved conversion in PF-CHO media with Efficient Feed C, including theaddition of glucose or galactose.

Addition of 5 μM or 20 μM Cu²⁺ at Day 0 had the same effect onconversion as 50 μM, as shown in FIG. 2. Addition of 50 μM Cu²⁺ at Day 3or Day 5 had the same effect on conversion as addition on Day 0. Cu²⁺(50 μM D0) improved conversion under culture conditions that includedPF-CHO, amino acids, AGT CD 5× medium, CellBoost4, glucose feed, andtemperature shift.

The experiments described above demonstrated that culturing cells in thepresence of Cu²⁺ resulted in FGE activation and, in turn, increasedconversion of the FGE recognition site in a target protein to include anFGly.

Experiments were also performed to test the effect on conversion inpresence of various concentrations of other ion sources, such asiron(II) sulfate, MnCl₂, and ZnCl₂. The experimental conditions were thesame as those used in the Cu²⁺ experiments above. The results indicatedthat iron sulfate (0.1 mM or 0.5 mM Day 3), MnCl₂ (50 uM or 10 uM Day0), and ZnCl₂ (50 uM Day 0) did not effect a detectable or significantincrease in conversion of the target polypeptide.

FIG. 3, Panel A shows data comparing the viable density of Cu²⁺-treatedcells and untreated cells. FIG. 3, Panel B shows data comparing theprotein titer of Cu²⁺-treated cells and untreated cells. FIG. 3, Panel Cshows data indicating in vivo FGE activation/increased conversion inCu²⁺-treated cells. The data shown in FIG. 3 indicate that the titer andcell viability does not significantly change in the presence of Cu²⁺ ascompared to experiments where no Cu²⁺ was added.

Example 2: The Effect of Potential Stimulators of Lactate Consumption onConversion Efficiency In Vivo

As copper is a potential stimulator of lactate consumption, the abilityof other potential stimulators of lactate consumption to activate FGEfor increased conversion efficiency of the FGE recognition site in atarget protein to include an FGly was investigated.

Experimental conditions were the same as those described in Example 1above. Addition of lactate (Sigma) or pyruvate (Sigma) (1.5 g/L withfeeds) to the cell culture medium did not increase conversion efficiencyof the FGE. PowerFeedA (Lonza), reported to stimulate lactateconsumption, did not increase conversion efficiency. Analysis of lactatelevels showed no differences in lactate consumption in the presence orabsence of Cu²⁺, in spite of differences in conversion efficiency in thepresence or absence of Cu²⁺.

FIG. 7, Panel A shows a graph of % conversion in the presence of Cu²⁺ orabsence of Cu²⁺, which indicated a higher % conversion in the presenceof Cu²⁺. FIG. 7, Panel B shows a graph of lactate consumption in thepresence or absence of Cu²⁺, which indicated no significant differencein lactate consumption in the presence or absence of Cu²⁺. FIG. 7, PanelC shows a graph of glucose consumption in the presence or absence ofCu²⁺, which indicated no significant difference in glucose consumptionin the presence or absence of Cu²⁺.

Example 3: Cu²⁺ Reduces Variability in Conversion Efficiency in a 293Expi System

A 293 Expi transient co-transfection system (Life Technologies) was usedfor producing antibodies having a formylglycine residue at a selectedsite. The system produced high antibody titers (100-500 mg/L) withvariable (10-95%) conversion. To determine whether the addition of Cu²⁺in the culture medium could reduce the variability in this system, 50 μMcopper sulfate was added just prior to transfection of CT-1.1 taggedHer2 or the day following transfection. Expi 293F that were treated withor without 50 μM copper sulfate prior to transfection were transientlyco-transfected in three independent experiments with expression plasmidsfor full-length human FGE with an N-terminal KDEL amino acid sequence(+KDEL) and CT tagged antibody at a ratio of 3:1 antibody:FGE DNA. Theculture was harvested at 5-6 days post transfection, titer assessed byELISA, and the protein was purified and conversion was measured bymass-spectrometry.

The results are shown in FIG. 4 and Table 1 and indicate that Cu²⁺enhances conversion in 293 cells without a significant reduction intiter.

TABLE 1 Copper Har- Conver- Experi- FGE sulfate Titer vest sion mentform (μM) Antibody (mg/L) day (%) Exp. 1 +KDEL 0 Her2 CT1.1 393 5 47+KDEL 50 μM Her2 CT1.1 418 5 95 at Day 0 Exp. 2 +KDEL 0 Her2 CT1.1 540 578 +KDEL 50 μM Her2 CT1.1 426 5 93 at Day 0 Exp. 3 +KDEL 0 Her2 CT1.1137 6 72 +KDEL 50 μM Her2 CT1.1 119 6 94 at Day 0

Example 4: Cu²⁺ Reduced Variability in Conversion Efficiencies ofVarious Forms of FGE

Variable conversion efficiency of the +KDEL form of human FGE may bereduced by addition of Cu²⁺. Using the same experimental conditions asdescribed above in Example 3, experiments were performed to determinethe ability of Cu²⁺ to reduce variability in conversion efficiency ofnon-KDEL forms of human FGE. Expi 293F cells that were treated with orwithout 50 μM copper sulfate prior to transfection were transientlyco-transfected with expression plasmids for various forms of human FGE(+KDEL, WT, myc-His) and CT tagged antibody at a ratio of 3:1antibody:FGE DNA. The culture was harvested at 5 days post transfection,titer assessed by ELISA, the protein was purified and conversion wasmeasured by mass-spectrometry.

The results are shown in FIG. 8 and Table 2 and indicated that Cu²⁺reduced variability in conversion efficiency in the Expi system whennon-KDEL forms of human FGE were co-transfected with tagged antibody,specifically WT FGE and FGE C-terminally tagged with myc-His.

TABLE 2 Copper Har- Conver- Experi- FGE sulfate Titer vest sion mentform (μM) Antibody (mg/L) day (%) Exp. 1 +KDEL 0 Her2 CT1.1 351 5 62+KDEL 50 μM Her2 CT1.1 232 5 88 at Day 0 WT 0 Her2 CT1.1 106 5 77 WT 50μM Her2 CT1.1 101 5 86 at Day 0 myc His 0 Her2 CT1.1 180 5 24 myc His 50μM Her2 CT1.1 124 5 78 at Day 0 Exp. 2 myc His 0 Her2 CT1.1 75 5 71 mycHis 50 μM Her2 CT1.1 64 5 94 at Day 0

Example 5: Effect of Stimulators and Inhibitors of OxidativePhosphorylation on Conversion

Experiments were performed to determine the effect of stimulators andinhibitors of oxidative phosphorylation on conversion efficiency forhuman FGE, as compared to Cu²⁺.

Dichloroacetate (DCA) is a pyruvate dehydrogenase kinase inhibitor thatstimulates mitochondrial respiration. Rotenone is a mitochondrialcomplex 1 inhibitor and inhibits oxidative phosphorylation. A doseresponse for ATP production with both agents (4 hour treatment) wasperformed and confirmed that each drug affected ATP production asexpected. Several experiments were run treating cells with DCA atseveral concentrations (DCA: 0 μM; 16 μM; 80 μM; 400 μM; 2000 μM; 10,000μM; and 50,000 μM) and treating cells with Cu²⁺ (copper sulfate) androtenone (Rotenone: 0 nM; 5 nM; 24 nM; 120 nM; 600 nM; 3000 nM; and15,000 nM) at several concentrations. Cells were plated and 10 μL of DCAor rotenone was added for a 4 hour treatment. Cells were equilibrated toroom temperature 100 μL/well of Cell titerglo reagent (Promega) wasadded to each well. The plates were shaken of 2 min, incubated at roomtemperature for 10 min, and then read on a plate reader.

The results indicated that there was no effect on conversion with eitherDCA or rotenone. The DCA treated cells exhibited slightly lowerconversion than untreated cells, and cells treated with rotenone andCu²⁺ had similar conversion efficiencies as cells treated with Cu²⁺alone. These results indicated that oxidative phosphorylation was likelynot involved in the enhanced conversion observed with Cu²⁺ treatment.

Example 6: Effect of Sulfide Quinone Reductase (SQR) on Conversion

Hydrogen sulfide (H₂S) is a putative side product of the FGE catalyticcycle. Sulfide quinone reductase (SQR) is a mitochondrial enzyme thatoxides and detoxifies H₂S. Experiments were performed to determinewhether H₂S accumulation over the course of the fed batch inhibitedhuman FGE activity, and if Cu²⁺ counteracted this effect.

Cells were plated. DNA was mixed with Optipro serum free medium (LifeTechnologies). FreeStyle Max transfection reagent (Life Technologies)and Optipro serum free medium were mixed. DNA and reagents were combinedfor 10-20 min at room temperature. The resulting complexes were addedthe cells.

A Western blotting was performed on lysates from cells treated with Cu²⁺or not treated with Cu²⁺, using antibodies against SQR to determinewhether Cu²⁺ increased the levels of SQR. No significant difference inthe levels of SQR was observed. Experiments were also performed to testwhether overexpression of SQR in Expi cells could be a substitute forCu²⁺ in increasing conversion. The results indicated that there was nosignificant difference in conversion in SQR overexpressing cellscompared to the control. SQR overexpression was confirmed in a CHOtransient transfection. Thus, SQR is not likely involved in maintaininghigh conversion in the stationary phase.

Example 7: FGE Isolated from Cu²⁺-Treated Cells has Increased SpecificActivity

To determine whether the enhanced conversion observed in Cu²⁺ treatedcells was due to increased specific activity of the FGE enzyme, Expicells were treated with and without Cu²⁺ and transfected with humanFGE-mycHis (pRW529). FGE was purified via nickel chromatography andtested for specific activity, with the FGE isolated from Cu²⁺ treatedcells consistently exhibiting significantly higher activity.Experimental conditions were the same as in Example 3 above. Specificactivity was measured as described in Example 9 below.

Providing Cu²⁺ in the cell culture medium resulted in activation of theFGE, as indicated by the higher specific activity of FGE isolated fromthe treated cells as compared to cells cultured in the absence of copperbut otherwise under identical conditions. As shown in FIG. 9, Panel A,and FIG. 9, Panel B, levels of FGE were the same (or lower with copper;see FIG. 9, Panel A) but the activity of FGE from copper cultures wassignificantly higher (see FIG. 9, Panel B).

Example 8: FGE Activation and Enhanced Conversion in Prokaryotic Cells

The ability of Cu²⁺ to activate FGE and enhance conversion inprokaryotic cells was investigated. E. coli cells that co-express S.coelicolor (“Sc”) FGEs and proteins that include FGE recognition siteswere cultured in the presence or absence of Cu²⁺. As shown in FIG. 4,conversion was enhanced in the cells treated with Cu²⁺ as compared tothe cells cultured in the absence of Cu²⁺.

To investigate the potential mechanism of FGE activation by Cu²⁺, liquidchromatography-mass spectrometry (LCMS) was carried out on Sc-FGEsisolated from E. coli cells cultured in the presence or absence of Cu²⁺.As shown in FIG. 5, Cu²⁺ treatment resulted in the oxidation of the twoactive site thiol groups of the Sc-FGEs, indicating that FGE activationmay involve disulfide formation involving the two active site cysteineresidues.

Example 9: In Vitro FGE Activation

The present example demonstrates that FGEs may be activated in vitro bytreatment with CuSO₄, resulting in treated/activated FGEs having greaterspecific activity as compared to untreated/non-activated FGEs.

Recombinant Expression and Purification of Sc FGE from E. coli Cells

Enhanced recombinant expression and purification of S. coelicolor (Sc)FGE was performed. A pET151-D/TOPO vector encoding the wild-type Sc-FGEcarrying an N-terminal hexahistidine tag and TEV protease cleavage sitewas transformed by heat shock at 42° C. into 50 μL ultracompetent E.coli BL21 (DE3) cells in LB medium. After plating and overnight growthon LB/agarose at 37° C. under ampicillin selection, individual colonieswere amplified to 125 mL in terrific broth (TB). At OD 0.4-0.5, IPTG wasadded to reach 1 mM. The culture flasks were cooled to 18° C. and shakenat 200 RPM for 16 h. Cells were collected at 6,000 rcf for 20 min. Cellswere resuspended in lysis buffer (25 mM triethanolamine, 50 mM NaCl,protease inhibitors, pH 8.0) and lysed with BugBuster® lysis reagent.The solutions were clarified by centrifugation at 25,000 rcf for 25 min.Residual DNA was precipitated by diluting the supernatant into asolution of streptomycin sulfate, reaching a final concentration of 1%w/v. The solution was stirred for 15 min at 4° C., and then centrifugedfor 25,000 RPM for 25 min. The supernatant was loaded onto a Ni-NTAsepharose FF column and eluted under gravity flow. Nonspecifically boundprotein was removed by washing with 5 CV of wash buffer (25 mMtriethanolamine, 250 mM NaCl, 10 mM imidazole, pH 8). The purifiedprotein was isolated with elution buffer (25 mM triethanolamine, 250 mMNaCl, 300 mM imidazole, pH 8). Fractions containing protein (as judgedby Bradford assay) were pooled and loaded onto a Sephadex® G-25 column(PD-10, GE Healthcare) equilibrated with storage buffer (25 mMtriethanolamine, 50 mM NaCl, 8% v/v glycerol, pH 7.4). The protein wasthen eluted with storage buffer, concentrated with a 10 kD Amicon®ultrafiltration membrane (Millipore, Inc.), and flash frozen at 77 K.Typical yield of isolated enzyme: 50-75 mg/L media. Purified enzyme wascharacterized by SDS-PAGE electrophoresis (10% gel, Bio-Rad, Inc.),reversed-phase HPLC, LCMS of the intact globular protein, LCMS of atryptic digest of the enzyme, UV-Vis absorption spectroscopy, specificactivity (as described below).

FGE Activity Assay

The specific activity of FGE was measuring using a discontinuousenzymatic activity assay. The substrate for FGE was a 14 amino acidpeptide containing the consensus sequence (H2N-ALCTPSRGSLFTGR-COOH). Therate of reaction was determined by integrating the peak area at 215 nmof the starting material (Cys) and product (fGly) containing peptides onreversed-phase HPLC. The identity of product (and side product) peakswere determined by RP-HPLC MS/MS. Each aqueous reaction solution (60 μL)contained substrate peptide (100 μM), FGE (1 μM), DTT (1 mM), and buffer(25 mM triethanolamine, pH 9). A single time course consisted of 5 datapoints collected every 2 minutes. The reaction was initiated uponaddition of FGE stock solution and vortexing for 3 s. Time points werequenched by diluting 10 μL of reaction mixture into 1 μL of 1 M HCl byhand with a micropipettor. After completion of the reaction, each timepoint was analyzed by RP-HPLC.

Quantification of FGE Substrate and Product Peptides by HPLC

Substrate, product, and side products of the FGE substrate peptideALCTPSRGSLFTGR were separated on RP-HPLC over 7 min with isocratic 18%MeCN in water containing 0.1% TFA. The integrated area of the fGly (2.1min), Cys (3.3 min), βME-DS (3.6 min), and Cys-DS (6.2 min) forms of thesubstrate were used to calculate total area and each fraction thereof.

Activation of FGE

To a solution of formylglycine-generating enzyme (S. coelicolor, 50 μM)in buffered aqueous solution (25 mM triethanolamine, pH 7.4, 50 mM NaCl)was added copper(II) sulfate (100 μM). The mixture was vortexed at 1500RPM for 1 h at 25° C. The protein was then removed from the reactionmixture by buffer exchange into 25 mM triethanolamine, pH 7.4, 50 mMNaCl using Sephadex® G-25 resin (GE healthcare PD-10 column followingmanufacturer's instructions). After exchange, the specific activity ofFGE was measured using the standard activity assay described herein.

Results

The results of the in vitro activation experiment are shown in FIG. 6,which shows a graph of the specific activities of FGE treated with CuSO₄as a source of Cu²⁺, and untreated FGE. Treatment with Cu²⁺ resulted inFGE activation, as indicated in this example by the increased specificactivity of the treated FGE.

Example 10: Production of Recombinant Baculovirus Encoding for the Coreof Human FGE

DNA encoding the catalytic “core” of H. sapiens FGE followed by aC-terminal His₆ tag for purification (Hs-cFGE) was amplified from aprevious construct (see Rabuka, et al., Nat. Protocols., (2012), 7,1052-1067) by PCR. The Hs-cFGE sequence and primers are listed below.Preparation of the Hs-cFGE-encoding baculovirus was performed using theBac-to-Bac baculovirus expression system (Invitrogen).

Primers Used for Cloning Hs-cFGE for the Bac-to-Bac Expression System.

Forward: GAGGCTAACGCTCCGGGCCC Reverse: GTCCATAGTGGGCAGGCGGTC

The Bac-to-Bac baculovirus expression system results in an additionalfour residues at the N-terminal end to provide a signal sequence ofamino acid sequence DRSL (SEQ ID NO:6). The primary sequence of Hs-cFGEas expressed from this expression vector is provided below.

Primary sequence of Hs-cFGE with TEV protease site and His₆ tag         10         20         30         40         50         60DRSLEANAPG PVPGERQLAH SKMVPIPAGV FTMGTDDPQI KQDGEAPARR VTIDAFYMDA        70         80         90        100        110        120YEVSNTEFEK FVNSTGYLTE VAAAPWWLPV KGANWRHPEG PDSTILHRPD HPVLHVSWND       130        140        150        160        170        180AVAYCTWAGK RLPTEAEWEY SCRGGLHNRL FPWGNKLQPK GQHYANIWQG EFPVTNTGED       190        200        210        220        230        240GFQGTAPVDA FPPNGYGLYN IVGNAWEWTS DWWTVHHSVE ETLNPKGPPS GKDRVKKGGS       250        260        270        280        290YMCHRSYCYR YRCAARSQNT PDSSASNLGF RCAADRLPTM DKGENLYFQG HHHHHH

Example 11: Expression and Purification of Hs-cFGE from Hi5 Cells

Hs-cFGE baculovirus was transfected into Hi5 insect cells (Invitrogen)at an MOI of 0.1. After 72 h in culture, the conditioned media wasclarified by centrifugation and filtration (0.45 μm PES), and loaded byFPLC onto HisTrap® Excel resin at 5 mL/min. The resin was washed with 10column volumes (CVs) of wash buffer (25 mM TEAM, 250 mM NaCl, 5 mMcalcium acetate, 10 mM imidazole, pH 8). The enzyme was then removedfrom the column with elution buffer (25 mM triethanolamine (TEAM), 5 mMcalcium acetate, 300 mM imidazole, pH 8). Fractions containing protein(as determined by UV detection of the FPLC elution) were pooled andloaded onto a Sephadex® G-25 column equilibrated with storage buffer (25mM TEAM, 8% v/v glycerol, pH 7.4). Then, the protein was eluted withstorage buffer, concentrated with a 10 kD Amicon® ultrafiltrationmembrane (Millipore), and flash frozen in liquid nitrogen. The typicalyield of isolated enzyme was 10-20 mg/L media. Purified enzyme wascharacterized by SDS-PAGE electrophoresis (FIG. 10), reversed-phaseHPLC, LC/MS of the intact protein, tryptic digestion followed byLC-MS/MS, absorption spectroscopy, and specific activity (as describedin the Examples below).

Example 12: Assay of FGE Catalytic Activity

The specific activity of FGE was measured using a discontinuousenzymatic activity assay. The substrate for FGE was a 14 amino acidpeptide containing the CXPXR consensus sequence (ALCTPSRGSLFTGR). Therate of reaction was determined by integrating the peak areas at 215 nmof the substrate cysteine (C_(sub)) and product (fGly) peptides onreversed-phase HPLC. The identities of product (and side product) peakswere determined by RP-HPLC MS/MS (see below). Each aqueous reactionsolution (120 μL) contained C_(sub) (100 μM), FGE (0.1-1 μM), DTT (1mM), and buffer (25 mM TEAM, pH 9). A single time course included 5 datapoints collected at evenly spaced intervals. The reaction was initiatedupon addition of FGE stock solution and vortexing for 3 s. The reactionvial was vortexed at 1,500 RPM and 25° C. in a thermomixer R(Eppendorf). Time points were hand quenched by adding 20 μL of reactionmixture to 2 μL of 1 M HCl using a micropipettor. After completion ofthe reaction, each time point was analyzed by RP-HPLC (see below).

Example 13: Identification of FGE Peptide Intermediates by LC-MS/MS andRP-HPLC

To identify the intermediates and products of in vitro conversion by FGEon C_(sub), reaction mixtures from the FGE activity assay were quenchedand analyzed by LC/MS. The reaction mixture included 0.5 mM peptide, 0.5μM human FGE, 5 mM 2-mercaptoethanol (βME), and 25 mM TEAM, pH 9 in atotal volume of 200 μL. Time points were prepared by adding 20 μL ofreaction mixture to 2 μL of 1 M HCl. The gradient was 5-25% MeCN inwater with 0.1% formic acid over 10 minutes. The relative intensitiesand retention times of the four peptide peaks detected at 215 nm onRP-HPLC were replicated in the LC-MS/MS data, allowing assignment ofeach peak by mass. The calculated and observed m/z ions, and assignmentsfor each peptide species, are shown in FIG. 11, FIG. 12, and FIG. 13.

Example 14: Quantification of FGE Substrate and Product Peptides by HPLC

Substrate, product, and side products of the FGE substrate peptideALCTPSRGSLFTGR were separated on RP-HPLC over 7 min with isocratic 18%MeCN in water containing 0.1% TFA. The integrated areas of the fGly (2.1min), C_(sub) (3.3 min), the C_(sub)/βME disulfide (C_(sub)-βME, 3.6min), and substrate/substrate disulfide (C_(sub)-C_(sub), 6.1 min) formsof the substrate were used to calculate total area and each fractionthereof.

Example 15: Thiol and Disulfide Mapping of FGE

To determine the oxidation state of thiol residues on either Sc-FGE orHs-FGE, a procedure was developed for mapping thiols and disulfides byLC/MS using sequential labeling, digestion, and orthogonal labeling.Step 1, initial labeling and tryptic digestion: the reaction mixture wascomposed of FGE (7.5 μg), NH₄HCO₃ (50 mM), iodoacetamide (20 mM), andtrypsin (0.75 μg) in a total volume of 30 μL. The tube containing thesample was incubated at 37° C. for 16 h. Step 2, quenching: water (6 μL)and DTT (4.8 μL of 0.5 M, 50 mM final concentration) were added to thereaction mixture from step 1. The tube containing the sample wasincubated at 37° C. and 1500 RPM for 1 h. Step 3, second labeling:N-ethylmaleimide (7.2 μL of 0.5 M, 150 mM final concentration) was addedto the reaction mixture from step 2. The tube containing the sample wasthen incubated at 37° C. and 1500 RPM for 1 h. After step 3, thereaction mixture was quenched with HCl (4.8 μL of 1 M, 100 mM finalconcentration). Digested peptides were separated with a gradient of0-50% MeCN in water with 0.1% formic acid over 15 min.Cysteine-containing peptides were detected by MRM/MS monitoringiodoacetamide, N-ethylmaleimide, and oxidative modifications of Cysresidues.

Example 16: Activation of FGE with Copper(II) Sulfate

Copper sulfate (50 μM, 5 molar equiv) was added to a solution of Sc-FGE(10 μM) in buffered aqueous solution (25 mM TEAM, pH 7.4, 50 mM NaCl).The mixture was vortexed at 1500 RPM for 1 h at 25° C. The protein waspurified from the reaction mixture by buffer exchange into 25 mM TEAM,pH 7.4, 50 mM NaCl using Sephadex® G-25 resin. Then, the specificactivity of FGE was measured using the activity assay described above.

Example 17: Quantification of fGly Content in Intact mAb

DTT (20 mM) was added to a solution of mAb (20 μg) in buffered aqueoussolution (25 mM NH₄HCO₃) to a final volume of 20 μL. The tube containingthe sample was incubated at 37° C. for 15 min at 1500 RPM. Then, HCl (50mM) was added to the solution and the tube was vortexed until mixed.Next, ammonium bicarbonate (100 mM) was added to the solution and thetube was vortexed until mixed. Trypsin (1 μg) and iodoacetamide (50 mM)were added, and the tube was incubated at 37° C. and 1500 RPM for 60 minin the dark. Finally, sodium citrate (150 mM, pH 5.5) and methoxylamine(100 mM) were added to the solution, and the tube was incubated at 37°C. for 16 h. The processed sample was then analyzed by LC-MRM/MS. TheMRM transitions included modifications of the Cys-containing peptideincluding: cysteine, cysteine modified with iodoacetamide,formylglycine, formylglycine hydrate (including loss of water in thecollision cell), and formylglycine methyl oxime. The abundance of eachspecies was defined by its 5 most intense precursor/product ion pairs,which were integrated to give the total signal and relative fractions ofeach component. In the optimized digestion and capping proceduredescribed above, unreacted Cys, fGly, or fGly-hydrate above backgroundwas not detected, observing only carboxyacetamidomethyl Cys (CAM) andthe methyl oxime of fGly (MeOx). Carboxyacetamidomethyl Cys (CAM) andthe methyl oxime of fGly (MeOx) were detectable.

Example 18: In Vitro Conversion of Intact mAb with FGE

Hs-cFGE was added to a solution of IgG containing the aldehyde tag(where the C-terminal K was substituted with SLCTPSRGS) in 25 mM TEAM pH9.0 with 50 mM NaCl and 1 mM βME. The enzyme was added at 10 mol %relative to the concentration of C_(sub) within the aldehyde tag of theantibody heavy chain. The solution was vortexed at 1,000 RPM for 16 h at18° C., and then the solution was quenched with 0.25 vol equiv of 0.5 Msodium citrate, pH 5.5 (to a final concentration 100 mM citrate) todecrease the pH of the solution to ˜7 for binding to mAb Select proteinA resin. The IgG was purified using standard methods. A typical reactionyielded a final fGly content in the antibody of 95-100%, correspondingto a reaction conversion yield of 90-100%. To date, the reaction hasbeen performed across a range of scales from 20 μg to 0.6 g.

Example 19: Generation of FGE Apoenzyme

Ethylenediaminetetraacetic acid (EDTA, 15 mM) andtris(carboxyethyl)phosphine (TCEP, 15 mM) were added to a solution ofSc-FGE holoenzyme (5 μM) in buffered aqueous solution (25 mM TEAM, pH7.4, 50 mM NaCl). The mixture was vortexed at 750 RPM for 1 h at 37° C.The protein was purified from the reaction mixture by buffer exchangeinto 25 mM TEAM, pH 7.4, 50 mM NaCl using Sephadex® G-25 resin. Then,the specific activity of FGE was measured using the activity assaydescribed above.

Results

The results from Examples 10-19 are discussed in the following sections.

Sc-FGE and Hs-cFGE had modest catalytic activity as isolated from cellculture. Methods for recombinant expression of FGE were developed. FGEsfrom two species—S. coelicolor and H. sapiens—were selected for studybecause they represented both prokaryotic and eukaryotic forms of theenzyme. Sc-FGE was prepared by transfection of E. coli and purificationfrom clarified cell lysates. Hs-cFGE was prepared in high purity andgood yield by viral transduction of insect cells. The human form wasproduced in insect cells.

With respect to the human enzyme, a noncatalytic portion of the enzymewas removed that appeared to facilitate precipitation (described indetail below). With soluble enzyme, the activity of FGEs as producedfrom cell culture was evaluated, and the reaction conditions requiredfor catalysis.

To measure the specific activity of FGE, a discontinuous assay wasperformed that used MALDI-MS to quantify the starting material andproduct of quenched reaction mixtures. In the assay, reversed-phase HPLCwas used both to separate and to quantify the Cys and fGly forms of apeptide containing the FGE consensus sequence. The unmodified 14-aminoacid peptide ALCTPSRGSLFTGR was used as a substrate for both the Sc- andHs-forms of the enzyme.

The conversion of Cys to fGly used a 2H⁺/2 e⁻ oxidation of the substrateCys thiol as well as exchange of sulfur for oxygen. Current data supporta mechanism in which molecular oxygen is the terminal electron acceptor.However, because O₂ is a 4H⁺/4 e⁻ acceptor, a reducing agent was used toprovide a second equivalent of 2H⁺ and 2 e⁻, completing the reductionfrom O₂ to 2 mol H₂O. To test if one might be able to bypass use of areductant by adding a 2H⁺/2 e⁻ oxidant directly to the in vitroreaction, the oxidant H₂O₂ was added, but resulted in dimerization ofthe substrate by generating a disulfide (Csub-Csub), which halted thereaction (FIG. 12, panel a and FIG. 12, panel b). A mixture of reductant(DTT) and O₂ was used for routine activity measurements.

Individual FGE reactions were initiated by rapidly mixing FGE into apremixed buffered solution of C_(sub) and reducing agent. It was assumedthat all buffered solutions contained oxygen at ˜270 μM at 25° C. (basedupon Henry's law). Time points were chemically quenched by hand withHCl. As described below, the specific activity of FGE can vary as afunction of the chemical state of its active site, and so enzymeconcentrations in this assay spanned the range of 0.1-10 μM (0.1-10 mol%) enzyme. The identities of both the Cys- and fGly-containing peptideswere confirmed by LC-MS/MS (FIG. 11). These assignments were thencorrelated with HPLC retention times (FIG. 14, panel a). The integratedpeak areas of substrate and product were used to calculate the initialvelocity of FGE (FIG. 14, panel b) using the method described by Boeker(Biochem. J., (1984), 223, 15; and Biochem. J., (1985), 226, 29).

Hs-FGE contains three primary domains: a signal peptide, an N-terminalextension (NTE), and a core that binds substrate and performs turnover(FIG. 14, panel c). After transport to the ER and proteolytic removal ofthe signal peptide, the “full length” wild-type enzyme (Hs-FGE) containsboth the NTE and the core. The NTE may facilitate ER retention throughdisulfide bond formation between C₅₀ and/or C₅₂ with ERp44. The borderbetween the NTE and the core is a proteolytic cleavage site forproprotein convertases such as furin and PACE. When FGE encounters theseenzymes in the secretory pathway, the NTE is removed and the truncatedcore can be secreted. In vitro, the full-length FGE had a propensity toaggregate at high concentrations, and as such the truncated core (cFGE)was prepared. The specific activity of Hs-cFGE (1,143 pkat·mg⁻¹, 33.3kD) was similar to Hs-FGE that contained the NTE (850 pkat·mg⁻¹, 36.9kD). In addition, Sc-FGE expressed in E. coli had a significantly lowerspecific activity (214 pkat·mg⁻¹), relative to the human enzymeexpressed in insect cells (FIG. 14, panel d). Thus, the experimentsshowed the production of active FGEs in good yield and high purity fromboth species. Further experiments were performed as described above toinvestigate the mechanism by which FGE performs catalysis and to enhancethe activity of FGE.

In some instances, the activity of FGE depended upon the presence of acopper cofactor, and not the redox state of active site cysteineresidues. Experiments were performed to test whether FGE requires thepresence of an active site disulfide residue. Reduced and oxidized formsof FGE were prepared and their specific activities were measured.

Formation of the reduced active site Cys residues was accomplished bypretreatment of Sc-FGE with an excess (100 molar equiv) of a strongreducing agent (DTT) for 1 h at 25° C., after which the reagent wasremoved by gel filtration. Formation of the oxidized active site wasaccomplished by pretreatment of the enzyme either with an excess (100molar equiv) of (L)-dehydroascorbic acid (DHAA) or CuSO₄ for 1 h at 25°C., followed by removal of the reagents by gel filtration. DHAA can formdisulfides stoichiometrically, and Cu²⁺ can rapidly, and cleanly,catalyze disulfide formation with dissolved oxygen. After thesetreatments, the specific activities of each enzyme were measured usingthe standard kinetic assay. Only pretreatment with CuSO₄ resulted in anysignificant change in activity (FIG. 15, panel a).

Since pretreatment and removal of copper led to more active FGE,experiments were performed to investigate the quantity of copper thatcould activate Sc-FGE. Treatment of FGE with 1, 10, or 100 mol % CuSO₄demonstrated that stoichiometric amounts of copper generated a highlyactive enzyme (FIG. 15, panel b). Amounts less than 1 molar equivresulted in proportionally lower activity, an observation that isinconsistent with the catalytic activation of active site thiols.

Experiments were performed to determine whether it is possible toenhance the FGE reaction rate by adding copper directly to the reactionmixture. The data confirmed that product formation was significantlyinhibited by the inclusion of copper in the reaction (FIG. 12, panel c).Rapid formation of the oxidized form of DTT (FIG. 12, panel d) as wellas C_(sub)-C_(sub) (FIG. 12, panel e) as a result of copper-catalyzeddisulfide formation was observed.

Both Sc-FGE and Hs-cFGE could be efficiently activated with 5 molarequiv of CuSO₄ for 1 h at 25° C. followed by removal of excess copper.The specific activity of Sc-FGE increased by more than an order ofmagnitude—from 214±34 pkat·mg⁻¹ to 3579±215 pkat·mg⁻¹; Hs-cFGE specificactivity increased by a factor of 3, from 1,143±84 pkat·mg⁻¹ to3,050±115 pkat·mg⁻¹ (FIG. 15, panel c). Experiments were performed todetermine whether FGE that was treated with copper contained the metalafter purification. The quantities of copper in both the unactivated andthe activated FGE samples were measured using ICP-MS. The Sc-FGEproduced in E. coli contained almost no copper above the level of thenoise of the measurement. By contrast, Hs-cFGE—as isolated from insectcells—contained 0.35 mol Cu per mol FGE. Both of the copper treated“activated” forms of FGE contained just above 1 mol Cu/FGE. Furthermore,the specific activity of each of these enzyme preparations correlatedwith the ratio of Cu/FGE, indicating that copper is an integralcomponent of the active form of FGE (FIG. 15, panel c).

It was then considered whether it is possible to enhance the FGEreaction rate by adding copper directly to the reaction mixture. Thedata indicated that product formation was significantly inhibited by theinclusion of copper in the reaction. Specifically, very rapid formationof the oxidized form of DTT as well as C_(sub)-C_(sub) was observed as aresult of copper-catalyzed disulfide formation (FIG. 15, panel d). Thevertical bars from left to right for each of fGly, C_(sub)-C_(sub) andDTT_(OX) correspond to Cu²⁺ concentrations of 0 μM, 5 μM and 50 μM.

To confirm that catalytic activity in the activated form of FGE did notdepend upon an active site disulfide, experiments were performed todetermine whether addition of strong reducing agents to the activatedenzyme would decrease the specific activity of the resulting material.If the integrity of the active site disulfide was important forturnover, then strong reducing agents should have decreased FGE turnoverrates.

Hs-cFGE may form the active site disulfide (C₃₃₆-C₃₄₁) as well as twostructural disulfides (C₃₄₆-C₂₃₅ and C₃₆₅-C₂₁₈) elsewhere in the protein(FIG. 16, panel a). Treatment of Hs-cFGE with 20 mM DTT (or 20 mM TCEP,not shown) produced an FGE with unchanged specific activity (FIG. 16,panel b). To confirm that the relevant Cys residues changed redox stateas a result of this treatment, an LC-MRM/MS assay was used to detectsolution-accessible Cys residues in the active site. With this method,both the C₃₄₁ ⁻ and C₂₃₅-containing peptides were monitored. Treatmentwith DTT increased the proportion of accessible active site C₃₄₁ from28% to 93%. C₂₃₅, which participated in a buried structural disulfide,was inaccessible (and thus, in a disulfide) independent of DTTtreatment. Taken together, these data confirmed that reductive treatmentgenerated almost quantitatively reduced active site thiols, did notperturb other structural disulfides, but had no effect on the catalyticactivity of FGE.

After observing that FGE contained ˜1 copper atom per enzyme,experiments were performed to remove the metal in order to determinewhether it was required for catalytic activity. Attempts to removecopper through the addition of 5 molar equiv EDTA did not change thecatalytic activity of copper-treated Sc-FGE or Hs-cFGE (FIG. 17, panela). KCN, however, did decrease the activity of Hs-cFGE by a modestamount, suggesting that it could gain access to the bound copper. Sincepretreatment with KCN slightly decreased the activity of Hs-cFGE,experiments were performed to test whether FGE turnover could beinhibited by inclusion of KCN in the reaction mixture. As shown in FIG.17, panel b, a concentration-dependent inhibition of activity on bothSc-FGE (IC₅₀=480 μM) and Hs-cFGE (88 μM) was observed. In the case ofcopper amine oxidases, apoenzyme has been generated by extracting copperwith KCN from enzyme that was pre-reduced with sodium dithionate. In asimilar manner, this approach was also successful for FGE. Activitydecreased significantly upon treatment (1 h, 37° C.) with both areductant and chelator (15 mM TCEP and EDTA), but not with eachcomponent individually (FIG. 17, panel d).

Experiments were performed to determine the standard enzymaticparameters of both Sc-FGE and Hs-cFGE (FIG. 18). They differedsignificantly between the two species. Sc-FGE did not interact withsubstrate very strongly (K_(M)=96 μM) and exhibited a relatively rapidk_(cat) (17.3 min⁻¹), which together resulted in a modest enzymeefficiency with k_(cat)/K_(M)=3.0×10⁴ M⁻¹·s⁻¹ (FIG. 18, panel 1). Bycomparison, Hs-cFGE turned over substrate with a slower catalytic rateconstant (k_(cat)=6.06 min⁻¹) but demonstrated a strong interaction withthe substrate peptide (K_(M)=0.34 μM). Taken together, the Hs-cFGE(k_(cat)/K_(M)=3.0×10⁶ M⁻¹0.5⁻¹) was approximately 10-fold moreefficient than its bacterial counterpart (FIG. 18, panel b).

The data demonstrated that the modestly active forms of FGE isolatedfrom cell culture were a mixture of the apoenzyme, lacking the coppercofactor, and the holoenzyme, which was highly active and efficientlyconverted Cys to fGly on peptide substrates. Experiments were performedto develop conditions for using the FGE holoenzyme in biocatalyticreactions on folded protein substrates.

FGE was an efficient biocatalyst for in vitro conversion of Cys to fGlyon folded proteins. In order to use in vitro conversion for theproduction of aldehyde-tagged mAbs, the following reaction conditionswere tested. For most substrates, 5-10 molar equiv of DTT was sufficientto achieve complete conversion. Turnover was also possible withtris(carboxyethyl) phosphine (TCEP), lipoic acid, andbis(2-mercaptoethyl)sulfone (BMS). However, the presence of even 1 molarequiv of a strong reducing agent (e.g., DTT, TCEP, lipoic acid, or BMS)could cleave disulfides that were present in a mAb substrate. To avoidsubstrate disulfide cleavage, an alternate reducing agent was used forthe reaction. Weaker reducing agents—e.g., βME or GSH—were tolerated byantibody substrates, and also enabled FGE catalysis. However, changingfrom a cyclizing (DTT) to a non-cyclizing reducing agent (βME) resultedin side products that had not previously been observed (discussedbelow). In order to reduce side product formation and increase productyield, experiments were performed to determine the role(s) of thereducing agent in enzymatic turnover.

X-ray crystallography of FGE (apoenzyme) bound to a substrate peptideshowed C₃₄₁ and C_(sub) covalently linked by a disulfide in the activesite. If the substrate was anchored by this C₃₄₁-C_(sub) disulfideduring turnover, an exogenous thiol might be able to react with theenzyme-substrate complex (E⋅S) by thiol-disulfide exchange to releasesubstrate in the form of a thiol— C_(sub) disulfide. Two off-pathwayproducts were formed by FGE during turnover. First, when reducing agentwas not added to the reaction, C_(sub)-C_(sub) dimer formation wasobserved. Second, when a monothiol was added as the reductant (e.g.,βME), rapid production of the C_(sub)-βME disulfide was observed (FIG.13, panel a). In control reactions lacking enzyme, neitherC_(sub)-C_(sub) nor C_(sub)-βME were generated.

The formation of C_(sub)-C_(sub) and C_(sub)-βME was monitored as afunction of reaction conditions. First, the concentration of reducingagent in the reaction was varied, and the reactions were quenched whenthey were half complete. At low [βME] v. [C_(sub)], the C_(sub)-C_(sub)predominated (FIG. 19, panel a). Increasing [βME] resulted in aconcurrent decrease in C_(sub)-C_(sub), and an increase in C_(sub)-βME.Increasing [βME] also resulted in a higher yield of fGly.

Experiments were performed to determine the effect of [βME] as thereaction evolved over time (FIG. 13, panel b). As described above, inthe absence of exogenous reducing agent product did form, but asignificant proportion of C_(sub)-C_(sub) was generated (FIG. 13, panelc), indicating that C_(sub) can act in place of the reducing agentduring turnover, but at the expense of product yield. At low [βME] (0.25mM, 2.5 molar equiv vs. C_(sub)), the C_(sub)-C_(sub) initially grew inat a rate competitive with product formation (FIG. 13, panel d), but asthe reaction proceeded, it reached a maximum, and then decayed awayslowly. At this [βME], the C_(sub)-C_(sub) was consumed, the C_(sub)-βMErapidly reached a plateau of 13% of total peptide, where it remained. Ina subsequent reaction with higher [βME] (10 mM), the consumption ofC_(sub)-C_(sub) was more rapid (FIG. 13, panel e). In addition, whileC_(sub)-βME quickly rose to 10% of the total substrate, it also decayedrapidly, in contrast to lower [βME]. In all cases, the consumption ofboth C_(sub)-C_(sub) and C_(sub)-βME correlated with a higher yield ofproduct formation. Since disulfide production during turnover wascompetitive with product formation, and did not occur in the absence ofenzyme, these data confirmed that disulfide formation between FGE andsubstrate was part of the FGE catalytic cycle.

In contrast to βME, when DTT was used as the stoichiometric reductantneither the intermediate C_(sub)-C_(sub) nor the C_(sub)-DTT disulfideswere observed. This was likely because DTT can cyclize to form anintramolecular disulfide and release substrate. However, the rates ofproduct formation in reaction using βME or DTT were not the same (FIG.19, panel b). Specifically, the rates of product formation were 5.07 or3.27 min⁻¹ in the presence of βME or DTT, respectively. Since reducingagent was present in large excess versus both the substrate and enzyme,it may be involved in limiting the rate of turnover.

If the reductants were acting by a one-electron mechanism, theirrelative reaction rates should depend directly upon their reductionpotentials:

E₀′(βME)=−207 mV and E₀′(DTT)=−323 mV. If instead, the reductants wereacting by thiol-disulfide exchange, their reaction rate should correlatewith their thiol-disulfide exchange rate constants: k_(βME)=1 andk_(DTT)=6×10⁵, as measured versus glutathione disulfide. Both of thesescenarios predict that reactions performed in the presence of DTT shouldhave a faster rate than those performed in the presence of βME. Bycontrast, reactions performed with DTT were slower than those with βME,and so the role of the thiol in the reaction may not depend on thereaction rate of the thiol alone.

The covalent disulfide in E⋅S that was confirmed by the experimentsabove may be the source of the difference in reaction rates describedabove (FIG. 19, panel c). From this state, E⋅S can either proceed toproduct with a rate constant of k_(cat), or it can react with anexogenous thiol by thiol-disulfide exchange with a rate constant ofk_(DS). For any given reducing agent, k_(cat) is likely the same basedupon the mechanisms of analogous copper-dependent oxidases, in whichsubstrate release, and not redox chemistry, is rate limiting. However,k_(DS) may depend directly upon the identity of the reducing agent insolution and its thiol-disulfide reaction rate constant. Said anotherway, E⋅S should dissociate faster in the presence of a stronger reducingagent, in competition with product formation. In addition, if thesubstrate was released as a disulfide that does not decay quickly (aswith βME-DS), it could react with the enzyme again to regenerate E⋅S.Therefore, stronger reductants should slow turnover by dissociating theE⋅S complex. This aspect of the cycle may also explain the fasterturnover rate observed at pH 9 relative to pH 7; thiol-disulfideexchange was faster under basic conditions.

The aldehyde tag can be introduced at the N- or C-terminus or at anysolvent accessible internal sequence. Here, Cys was converted to fGly inhigh yield by Hs-cFGE in three independent regions of a mAb (FIG. 20,panel a) and across a wide range of reaction scales (from 0.8-200 mg) onthree independent mAbs (FIG. 20, panel b, and FIG. 20, panel c). In eachcase, the in vitro conversion yield was >90%, as measured by LC-MRM/MS(FIG. 21). Furthermore, the pH 9 reaction conditions did not affectantigen binding affinity (FIG. 22, panel a, and FIG. 22, panel b) oroxidation at Met252 (FIG. 22, panel c), a residue that is important forFcRn binding and mAb circulation in vivo.

DISCUSSION

Methods for recombinant production of both Sc-FGE and Hs-cFGE in goodyield are described herein. In addition, a discontinuous HPLC assay tomeasure the kinetic parameters of both FGEs is described. These resultsindicated that the N-terminal extension of Hs-FGE was not required forcatalytic activity in vitro. As isolated from insect cell culture, thehuman enzyme was significantly more active than the bacterial form ofFGE from S. coelicolor as produced in E. coli.

Experiments were performed to determine the mechanism of FGE turnover.The existing mechanistic hypotheses focus on the redox state of the FGEactive site cysteine residues as revealed by crystallography. WhenHs-cFGE was isolated previously, C₃₃₆ (C₂₇₂ in Sc-FGE) and C₃₄₁ (C₂₇₇)were present in the enzyme in a mixture of reduced and oxidized forms.The oxidation state of C₃₃₆ and C₃₄₁ can be manipulated using chemicalreagents in solution, either to form the C₃₃₆-C₃₄₁ disulfide, or withH₂O₂ over a longer period of time, to form a sulfonic acid at bothpositions. In addition, a C₃₃₆5 mutation enabled the “trapping” ofC_(sub) as an intermolecular disulfide with C₃₄₁. This indicated thatFGE uses a thiol-disulfide interchange to anchor C_(sub) and activateC₃₃₆. This residue may then perform the activation of molecular oxygento form a higher oxidation state Cys residue (either sulfenic acid orcysteine peroxide) and subsequently oxidize the substrate.

FGE was able to catalyze the formation of disulfides at the substrateCys when monothiol reducing agents were present in solution. Thisindicated that FGE bound C_(sub) in a disulfide, and that when k_(cat)was slower than the rate of reaction with reducing agent in solution,C_(sub) can be released as a disulfide. When dithiol reagents such asDTT were used, any intermolecular disulfide was quickly abolished by DTTcyclization.

FGE incorporates copper as a cofactor for catalysis. The resultsindicated that recombinant expression of FGE most often resulted in theapoenzyme, which can be reconstituted as the holoenzyme by the additionof copper(II) sulfate at pH 7. The catalytic rates of both human andbacterial FGE were significantly increased upon activation withstoichiometric amounts of Cu²⁺, and turnover was inhibited by theaddition of cyanide to FGE reaction mixtures.

The data indicated that FGE may be a copper oxidase that performs 2H⁺/2e⁻ oxidation, activates molecular oxygen, and requires exogenousreductants to complete a catalytic cycle.

From the data described above, the formation of side products indicatedthat the [E⋅S] complex involved a covalent disulfide bridge (FIG. 23,step a). Reducing agent would serve to generate the Cu(I) state of theactive site (FIG. 23, step b), which is an intermediate for binding ofmolecular oxygen (FIG. 23, step c) as the cupric superoxo Cu(II)-O₂ ⁻that is poised for substrate oxidation. The substrate cysteine sidechaincould then react by proton-coupled electron transfer, most likely in theform of hydrogen atom transfer (FIG. 23, step d). After oxidation, thedisulfide radical may collapse to the thioaldehyde and a thiyl radical(FIG. 23, step e). The second equivalent of 1H⁺/1 e⁻ would regeneratethe active site Cys, and the thioaldehyde would rapidly hydrolyze to theproduct fGly (FIG. 23, step f and step g). Formally the oxidationreaction would be complete simply by displacing H₂O₂ as a reactionproduct, but the cupric peroxide Cu(II)-OOH might also undergo furtherreduction to generate only H₂O as a byproduct. At this point, theresting state ligand on copper may be a hydroxide, although directinvolvement of C₃₄₁ through an alternative mechanism may be possible.

As described above, reaction conditions were developed for efficientproduction of fGly without disrupting existing intramolecular disulfidesin the substrate protein. FGE was produced in good yield and used as abiocatalyst for the production of aldehyde-tagged proteins in vitro.Reactions with FGE scaled across at least three orders of magnitude inmass with no significant decrease in yield, and proceeded withoutperturbing existing disulfides or modifying residues that were importantfor mAb performance in vivo.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

1. A method of producing a protein comprising a formylglycine residue,the method comprising: combining an activated formylglycine-generatingenzyme (FGE) with a protein comprising an FGE recognition site underconditions in which the activated FGE converts a cysteine residue or aserine residue of the FGE recognition site to a formylglycine residue,to produce a protein comprising a formylglycine residue.
 2. The methodaccording to claim 1, wherein the combining comprises: culturing a cellcomprising: a formylglycine-generating enzyme (FGE); and the proteincomprising the FGE recognition site, in a cell culture medium comprisingCu²⁺ under cell culture conditions in which the FGE converts thecysteine residue or the serine residue of the FGE recognition site tothe formylglycine residue.
 3. The method according to claim 2, whereinthe Cu²⁺ is present in the cell culture medium at a concentration offrom 1 nM to 10 mM.
 4. The method according to claim 3, wherein the Cu²⁺is present in the cell culture medium at a concentration of from 1 μM to1 mM.
 5. The method according to claim 2, wherein the FGE is endogenousto the cell.
 6. The method according to claim 2, wherein the cell isgenetically modified to express an FGE.
 7. The method according to claim2, wherein the protein containing an FGE recognition site is endogenousto the cell.
 8. The method according to claim 2, wherein the cell isgenetically modified to express the protein containing an FGErecognition site.
 9. The method according to claim 2, wherein the cellis a eukaryotic cell.
 10. The method according to claim 9, wherein theeukaryotic cell is a mammalian cell.
 11. The method according to claim10, wherein the mammalian cell is selected from the group consisting of:a CHO cell, a HEK cell, a BHK cell, a COS cell, a Vero cell, a Helacell, an NIH 3T3 cell, a Huh-7 cell, a PC12 cell, a RAT1 cell, a mouse Lcell, an HLHepG2 cell, an NSO cell, a C127 cell, a hybridoma cell, aPerC6 cell, a CAP cell, and a Sp-2/0 cell.
 12. The method according toclaim 10, wherein the mammalian cell is a human cell.
 13. The methodaccording to claim 9, wherein the eukaryotic cell is a yeast cell. 14.The method according to claim 9, wherein the eukaryotic cell is aninsect cell. 15.-27. (canceled)
 28. The method according to claim 1,wherein the protein is an antibody, an antibody fragment, a ligand, anenzyme, or an antigen.
 29. The method according to claim 1, wherein theprotein is an antibody or antibody fragment.
 30. The method according toclaim 29, wherein the antibody or antibody fragment is selected from thegroup consisting of: an IgG or fragment thereof, a Fab, a F(ab′)2, aFab′, an Fv, an ScFv, a bispecific antibody or fragment thereof, adiabody or fragment thereof, a chimeric antibody or fragment thereof, amonoclonal antibody or fragment thereof, a humanized antibody orfragment thereof, and a fully human antibody or fragment thereof. 31.The method according to claim 29, wherein the antibody specificallybinds to a tumor-associated antigen or a tumor-specific antigen.
 32. Themethod according to claim 31, wherein the tumor associated antigen ortumor-specific antigen is selected from the group consisting of: HER2,CD19, CD22, CD30, CD33, CD56, CD66/CEACAM5, CD70, CD74, CD79b, CD138,Nectin-4, Mesothelin, Transmembrane glycoprotein NMB (GPNMB),Prostate-Specific Membrane Antigen (PSMA), SLC44A4, CA6, and CA-IX. 33.The method according to claim 1, wherein the protein is a ligand. 34.The method according to claim 33, wherein the ligand is a growth factoror a hormone.
 35. (canceled)
 36. The method according to claim 1,further comprising conjugating an agent to the protein comprising theformylglycine residue via an aldehyde moiety of the formylglycineresidue.
 37. The method according to claim 36, wherein the agent is atherapeutic agent.
 38. The method according to claim 37, wherein thetherapeutic agent is selected from the group consisting of: a cytotoxicagent, an antiproliferative agent, an antineoplastic agent, anantibiotic agent, an antifungal agent, and an antiviral agent.
 39. Themethod according to claim 36, wherein the agent is an imaging agent. 40.(canceled)
 41. A composition comprising: a cell culture mediumcomprising Cu²⁺; and a cell present in the cell culture medium, whereinthe cell expresses formylglycine-generating enzyme (FGE). 42.-52.(canceled)
 53. A method comprising: culturing a cell that comprises anucleic acid encoding a formylglycine-generating enzyme (FGE) in a cellculture medium that comprises Cu²⁺, wherein the culturing is underconditions in which the FGE is expressed in the cell.
 54. The methodaccording to claim 53, wherein the Cu²⁺ is present in the cell culturemedium at a concentration of from 0.1 μM to 10 mM.
 55. (canceled) 56.The method according to claim 53, wherein the FGE is endogenous to thecell.
 57. The method according to claim 53, wherein the cell isgenetically modified to express an FGE.
 58. The method according toclaim 53, wherein the cell is genetically modified to express a proteincontaining an FGE recognition site.
 59. The method according to claim53, wherein the cell is a eukaryotic cell. 60.-63. (canceled)
 64. Themethod according to claim 53, wherein the cell is a prokaryotic cell.65. A method of producing an activated formylglycine-generating enzyme(FGE), comprising treating an FGE with Cu²⁺ to produce an activated FGE.66. The method according to claim 65, wherein treating the FGE with Cu²⁺comprises culturing a cell that comprises a nucleic acid encoding theFGE in a cell culture medium that comprises Cu²⁺, wherein the culturingis under conditions in which the FGE is expressed in the cell. 67.-70.(canceled)
 71. The method according to claim 66, wherein the cell is aeukaryotic cell. 72.-75. (canceled)
 76. The method according to claim66, wherein the cell is a prokaryotic cell.
 77. The method according toclaim 66, further comprising purifying the FGE from the cell. 78.-86.(canceled)
 87. An activated formylglycine-generating enzyme (FGE)produced by the method according to claim
 65. 88. A cell-freecomposition comprising: an activated formylglycine-generating enzyme(FGE); and a buffer. 89.-96. (canceled)